SuperScript Choice System for cdna Synthesis

Transcription

1 USER GUIDE SuperScript Choice System for cdna Synthesis Catalog number Document Part Number18090 Publication Number MAN Revision 3.0 For Research Use Only. Not for use in diagnostic procedures.

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3 Table of Contents 1. Notices to Customer Precautions Limited Label License No. 358: Research Use Only Trademarks Disclaimer Overview cdna Libraries mrna Isolation Choosing the Priming Method First-Strand Synthesis Second-Strand Synthesis Maximizing Ligation Efficiency by Adapter Addition Size Fractionation of cdna Choosing the Cloning Vector Plasmid Vectors λ Vectors Ligation of Size-Fractionated cdna to the Vector of Choice Methods Components General Comments mrna Purification Advance Preparations Time Planning Utilization of Reagents First-Strand Synthesis Second-Strand Synthesis EcoR I (Not I) Adapter Addition Phosphorylation of EcoR I-Adapted cdna Column Chromatography Ligation of cdna to a Plasmid Vector Ligation of cdna to λgt11 and λgt10 Vectors Ligation of cdna to a λziplox Vector Analysis of cdna Products First-Strand Yield Second-Strand Yield Gel Analysis Analysis of cdna from the cdna Size Fractionation Column Troubleshooting Isolation of mrna First-Strand Reaction Second-Strand Reaction EcoR I (Not I) Adapter Addition Phosphorylation of EcoR I-Adapted cdna...27 i

4 Table of Contents 4.6 Column Chromatography Ligation of cdna to a Plasmid Vector Ligation of cdna to a λ Vector References Related Products...30 Figures 1. Summary of the SuperScript Choice System Procedure Effect of Random Hexamer Concentration on First-Strand cdna Synthesis Sequence of the EcoR I (Not I) Adapter Detailed Protocol Flow Diagram Alkaline Agarose Gel Analysis of First and Second-Strand cdna Synthesized with the SuperScript Choice System Electrophoretic Analysis of Size-Fractionated cdna...25 ii

5 Notices to Customer Precautions Warning: This product contains hazardous reagents. Consult the applicable SDS(s) before using this product. Disposal of waste organics, acids, bases, and radioactive materials must comply with all appropriate federal, state, and local regulations. 1.2 Limited Label License No. 358: Research Use Only The purchase of this product conveys to the purchaser the limited, non-transferable right to use the purchased amount of the product only to perform internal research for the sole benefit of the purchaser. No right to resell this product or any of its components is conveyed expressly, by implication, or by estoppel. This product is for internal research purposes only and is not for use in commercial applications of any kind, including, without limitation, quality control and commercial services such as reporting the results of purchaser's activities for a fee or other form of consideration. For information on obtaining additional rights, please contact outlicensing@lifetech.com or Out Licensing, Life Technologies, 5791 Van Allen Way, Carlsbad, California Trademarks The trademarks mentioned herein are the property of Life Technologies Corporation or their respective owners. Sephacryl is a registered trademark of GE Healthcare Bio-Sciences. TRIzol is a registered trademark of Molecular Research Center, Inc. RNase Away and ART are registered trademarks of Molecular Bio-Products, Inc. 1.4 Disclaimer LIFE TECHNOLOGIES CORPORATION AND/OR ITS AFFILIATE(S) DISCLAIM ALL WARRANTIES WITH RESPECT TO THIS DOCUMENT, EXPRESSED OR IMPLIED, INCLUDING BUT NOT LIMITED TO THOSE OF MERCHANTABILITY, FITNESS FOR A PARTICULAR PURPOSE, OR NON-INFRINGEMENT. TO THE EXTENT ALLOWED BY LAW, IN NO EVENT SHALL LIFE TECHNOLOGIES AND/OR ITS AFFILIATE(S) BE LIABLE, WHETHER IN CONTRACT, TORT, WARRANTY, OR UNDER ANY STATUTE OR ON ANY OTHER BASIS FOR SPECIAL, INCIDENTAL, INDIRECT, PUNITIVE, MULTIPLE OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT, INCLUDING BUT NOT LIMITED TO THE USE THEREOF Life Technologies Corporation. All rights reserved. 1

6 2 Overview 2.1 cdna Libraries A cdna library is an array of DNA copies of an mrna population that are propagated in a cloning vector and usually maintained in E. coli. A good cdna library is large enough to contain representatives of all sequences of interest, some of which may be derived from low-abundance mrnas. Depending on the research objective, cdna library construction can begin by priming first-strand synthesis with oligo(dt) or with random hexamers. These two priming methods will ultimately produce two different types of cdna libraries, each of which will serve a different purpose. Libraries produced using oligo(dt) priming will contain cdna inserts of the largest size; libraries produced using random hexamer priming may contain a higher proportion of cdna with the 5 -most sequence information and may include cdna copies of poly(a) - as well as poly(a) + RNA. Careful planning and appropriate choices will result in cdna libraries tailored to specific research objectives. Because a cdna library is the end product of many individual steps, its quality can be compromised by inefficiency at any point in the procedure. The SuperScript Choice System integrates state-of-the-art cdna synthesis with simplified downstream technology to produce cdna that can be ligated to any EcoR I-digested vector for subsequent introduction into E. coli. If the starting mrna is of high quality, the cdna library constructed with this system will satisfy the preceding criteria. cdna libraries can be broadly classified as directional or random. Whereas all members of a directional library contain cdna inserts cloned in a specific orientation relative to the transcriptional polarity of the original mrnas, members of random libraries contain cdna inserts cloned in either orientation. For maximum versatility, the SuperScript Choice System has been designed for generation of random libraries: the double-stranded, EcoR I-ended cdnas produced with this system are suitable for insertion into the vast majority of existing vectors. The major steps in constructing a random cdna library from an mrna population using the SuperScript Choice System are summarized in Figure mrna Isolation Construction of a good cdna library begins with the preparation of high-quality mrna. The quality of the mrna dictates the maximum amount of sequence information that can be converted into cdna. Thus, it is important to optimize the isolation of mrna from a given biological source and to prevent adventitious introduction of RNases into a preparation that has been carefully rendered RNasefree. For optimal results, the mrna must be purified over an affinity column [oligo(dt) cellulose being the most commonly used matrix] to select the polyadenylated [poly(a) + ] RNA (1). Since the vast majority of mrna is poly(a) +, this selection operationally defines the mrna population. Typically, 0.5% to 2% of a total RNA population is mrna, so isolation of this fraction from rrna, trna, and degraded mrna enhances the synthesis of first-strand cdna and minimizes spurious transcription of non-mrnas. An mrna preparation that has undergone two selections over this matrix (in which the eluate from one round of purification has been bound to the column and eluted a second time) will produce the highest quality mrna. When properly prepared, oligo(dt) cellulose-purified RNA will be 90% mrna. 2

7 2 Figure 1. Summary of the SuperScript Choice System procedure. The amount of mrna needed to prepare a library is dependent on the efficiency of the individual steps needed to convert the mrna into a form that can be cloned and on the efficiency with which the recombinant molecules can be introduced into a host. Generally, 1 to 5 µg of mrna will be sufficient to construct a cdna library containing 10 6 to 10 7 clones in E. coli. 3

8 Overview 2.3 Choosing the Priming Method First-strand cdna synthesis is most commonly primed using oligo(dt) or modifications of this sequence (such as primer-adapters) that bind to the poly(a) tail of mrna. This priming method offers two major advantages: only poly(a) + RNA is copied, and most cdna clones begin at the 3 terminus of the mrna. At the same time, oligo(dt) priming has certain limitations: some cdna clones may not be full length, due to RNA secondary structure or pausing by reverse transcriptase, and poly(a) - mrna cannot be copied. An alternative method is to use random hexamers, which, in theory, are capable of binding and priming throughout virtually any RNA template. Random hexamers, which may be used either by themselves or in combination with oligo(dt), have been instrumental in producing cdnas containing more 5 information than those primed with oligo(dt) alone (2,3). In addition, random hexamers can be used to generate cdna libraries from poly(a) - mrna (4) and single-stranded viral RNAs (2,5). Although increasing the random hexamer concentration increases the percentage yield of first-strand cdna synthesis, it also decreases the average cdna size. A typical random hexamer titration profile (using cdna synthesized from HeLa mrna), as compared to the result obtained with oligo(dt) priming, is shown in figure 2. Identical random hexamer titration profiles are obtained whether the firststrand cdna reaction is incubated at 37 C or 45 C, using from 1 5 µg of mrna per reaction, and in the presence or absence of oligo(dt) primers. Note: A certain amount of self-priming by subpopulations of mrna may occur in the absence of any exogenous primers (see lane 2 of figure 2), which can contribute nominally (~6%) to the overall first-strand cdna yield produced by random hexamer priming Kb µg ,000 5,000 Figure 2. Effect of random hexamer concentration on first-strand cdna synthesis. 2 µg of HeLa mrna was primed with 1 µg of oligo(dt) (lane 1) or various amounts of random hexamers (lanes 2 through 9). 4

9 The SuperScript Choice System includes both oligo(dt) and random hexamer primers. In the decision to use either priming method separately, or both in combination, to synthesize cdna, the following considerations should be noted: 1. Priming with oligo(dt) by itself is decidedly better at producing larger cdna inserts. 2. Random hexamer priming yields cdna that are smaller on average but that may better represent the entire RNA template. Priming with random hexamers at a concentration of ng per reaction generally yields twice the amount of 5 -end information of the β-actin mrna as oligo(dt) priming (6). For some RNAs, however, higher concentrations of random hexamers may be needed to increase the proportion of cdna containing the 5 -most sequence information. 3. Random hexamers and oligo(dt), when used in combination, should be added simultaneously to ensure all possible priming events. 2.4 First-Strand Synthesis Avian myeloblastosis viral (AMV) reverse transcriptase (RT) was the first enzyme used to synthesize cdna in vitro, and much of the early work in cdna synthesis and cloning was developed using this enzyme. However, the successful cloning of the Moloney murine leukemia virus (M-MLV) RT (7) has provided researchers with an alternative enzyme. The cloned M-MLV RT gene has been further engineered to produce a novel enzyme (SuperScript II RT) with reduced RNase H activity (8). This modification is significant because RNase H activity is detrimental to the firststrand cdna synthesis reaction in two ways: 1. The initiation of first-strand synthesis depends upon the hybridization of a primer to the mrna, usually at the poly(a) tail. This hybrid is a substrate, not only for the polymerase activity of the RT but also for the RNase H activity (9). In the resulting competition between these two activities, the extent to which the RNase H activity destroys the hybrid prior to the initiation of polymerization determines the maximal number of initiation events that can actually occur. Hydrolysis of the RNA in the hybrid reduces the maximal yield of cdna by effectively removing a portion of the mrna from the reaction. 2. When the RT is synthesizing the first-strand cdna, the RNase H activity will quickly degrade the template that has already been copied because the mrna is in hybrid form as a result of the polymerization reaction. If the scissions in the mrna occur near the point of chain growth, the uncopied portion of the mrna can dissociate from the transcriptional complex, resulting in termination of cdna synthesis for that template and consequent reduction in the yield of full-length cdna. This problem can be exacerbated if the RT pauses during transcription at certain primary or secondary structural domains. When used with synthetic RNA produced in vitro, SuperScript II RT has demonstrated significantly greater full-length cdna synthesis and higher yields of first-strand cdna than other commercially available RTs (10, 11, 12). The reaction conditions for first-strand synthesis catalyzed by SuperScript II RT have been optimized for yield and size of the cdnas. The optimal first-strand reaction temperature for SuperScript II RT is 37 C; however, should secondary structure make reverse transcription difficult, a higher reaction temperature may be used. SuperScript II RT is stable at 45 to 50 C and can be used at this temperature, if necessary. The amount of mrna can be as high as 5 µg in a 20-µL first-strand cdna synthesis reaction. We recommend using at least 1 µg of mrna so that there will be sufficient material at the end of the procedure to obtain the required number of clones. The amount of SuperScript II RT needed in the first-strand reaction varies linearly with the amount of mrna: 200 units of SuperScript II RT for 1 µg of mrna, and 200 units per µg for 1 5 µg of mrna. Although the exact ratio of SuperScript II RT to mrna is not critical, these approximate proportions have produced reliable results. 2 5

10 Overview 2.5 Second-Strand Synthesis The primary sequence of the mrna is recreated as second-strand DNA using the first-strand cdna as a template. The SuperScript Choice System uses nick translational replacement of the mrna to synthesize the second-strand cdna. First described by Okayama and Berg (13), and later popularized by Gubler and Hoffman (14), second-strand synthesis is catalyzed by E. coli DNA polymerase I in combination with E. coli RNase H and E. coli DNA ligase. Although RNase H is not essential if the first-strand synthesis is catalyzed by AMV or M-MLV RT, E. coli RNase H must be included in the second-strand reaction when SuperScript II RT has been used for first-strand cdna synthesis. E. coli DNA ligase has been shown to improve the cloning of double-stranded (ds) cdna synthesized from longer ( 2 kb) mrnas (15). The first and second-strand syntheses are performed in the same tube without intermediate organic extraction or ethanol precipitation. This one-tube format speeds the synthesis procedure and maximizes recovery of cdna. The efficiency of the second-strand reaction is influenced by the amount and concentration of the reactants, so the instructions must be followed as described for best results. The second-strand reaction is incubated at 16 C to prevent spurious synthesis by DNA polymerase I due to its tendency to strand-displace (rather than nick translate) at higher temperatures. The last step in the cdna synthesis procedure is to ensure that the termini of the cdna are blunt. This is easily done by adding T4 DNA polymerase to the second-strand reaction mixture and incubating briefly at 16 C. The cdna is then deproteinized by organic extraction and precipitated with ethanol to render it ready for downstream manipulation. 2.6 Maximizing Ligation Efficiency by Adapter Addition The product of the first and second-strand synthesis reactions performed using the SuperScript Choice System is blunt-ended cdna, a poor substrate for T4 DNA ligase. To maximize ligation efficiency into the vector, the blunt ends of the cdna are converted to termini that contain 5 extensions by adding adapters to the cdna. Adapters are short, duplex oligomers, blunt-ended at one terminus and containing a 4-base, 5 extension at the other terminus. The blunt-end ligation of the adapters to the cdna can be driven by adapter excess, much the way molecular linkers are added to DNA. However, unlike linkers, adapters contain preformed extensions and do not require restriction digestion to expose the termini. The 4-base, 5 extension of the adapters provided with the SuperScript Choice System corresponds to the termini produced by digestion with EcoR I. The sequence of the EcoR I (Not I) adapter included in the SuperScript Choice System is shown in Figure 3. Several details should be noted: 1. The recognition sequences for Not I and Sal I are contained within the EcoR I (Not I) adapter to allow easy release of cdna inserts. Both restriction endonuclease sites are extremely rare in mammalian genomes, occurring approximately once in 10 6 bp (Not I) and 10 5 bp (Sal I). 2. Only one of the oligomers of the EcoR I (Not I) adapter is phosphorylated, which drives adapter-to-cdna ligation and essentially eliminates cdna-tocdna ligation. 6

11 5 -AATTCGCGGCCGCGTCGAC GCGCCGGCGCAGCTGp-5 EcoR I Not I Sal I 2 Figure 3. Sequence of the EcoR I (Not I) adapter. Adding the EcoR I (Not I) adapters to the cdna places the same EcoR I 5 extension at both ends of the cdna. The cdna is then used to construct a random library by ligating it to an EcoR I-digested vector that has been dephosphorylated to reduce the background arising from self-ligation of the vector. This, in turn, requires phosphorylation of the adapted cdna at its 5 termini so that it can be ligated to the 5 -dephosphorylated, EcoR I-digested vector. The ligation and phosphorylation steps used in the SuperScript Choice System are performed in the same buffer without any organic extraction or ethanol precipitation, which maximizes efficiency and facilitates cdna recovery. 2.7 Size Fractionation of cdna Size fractionation of cdna, following adapter addition and phosphorylation, is important because residual adapters are present in large molar excess and can impede vector ligation to cdna by ligating to the EcoR I termini of the predigested vector. Size fractionation also reduces the tendency of smaller (<500 bp) inserts to predominate the library. These smaller cdnas can arise for several reasons: 1. Most mrna preparations are not size-selected, so partially degraded mrnas can be selected on the oligo(dt) cellulose columns along with longer mrnas. These will be reverse transcribed into small cdnas. 2. If extreme care is not taken to prevent RNase contamination during first-strand synthesis, degradation can occur when the mrna is manipulated. 3. Some mrnas contain regions that are not readily reverse transcribed, and RT is not able to synthesize complete first strands. Column chromatography is the simplest method of producing size-fractionated cdna, free of adapters and other low molecular weight DNAs. The SuperScript Choice System contains three 1-mL, disposable columns, prepacked with Sephacryl S-500 HR, that quickly and easily remove DNAs <500 bp and sizefractionate cdnas >500 bp, as well as perform an exchange of buffers, thus facilitating construction of libraries from fractions enriched for larger cdna. The column chromatography buffer (described in Chapter 3) is formulated to allow the cdna in the column factions to be ligated directly into predigested plasmid cloning vectors. Although the final yield of size-fractionated cdna will depend upon the recovery at each step of the procedure, the average overall yield should be 5% 10% of the mass of the starting mrna. 2.8 Choosing the Cloning Vector Two types of vectors are generally used for cdna cloning in E. coli plasmids and bacteriophage lambda (λ) derivatives. Several factors affect the choice of vector. Ease of use. Cloning into plasmids is generally easier for the novice because it requires less manipulation of the cdna and avoids potential problems when propagating phage. However, this consideration alone should not preclude using a λ vector if it is otherwise the best choice. For detailed discussions of cdna cloning into λ vectors, see references 16 and 17. 7

12 Overview Antibody screening. λ vectors are the better choice if the cdna library is to be screened with the antibody (18). Although plasmid-based cdna libraries may also be screened by this method (19), the process is more cumbersome and considerably more tedious. Nucleic acid screening. Choosing between plasmid and λ vectors is less critical if the library will be screened with a nucleic acid probe because performance is similar in both systems (20,21). Other considerations (such as subcloning capability) may dictate which system to use. The SuperScript Choice System is designed to produce cdna containing EcoR I-cohesive ends suitable for ligation to any EcoR l-digested, dephosphorylated plasmid or λ vector (consult reference 17 for details on preparing dephosphorylated vectors) Plasmid Vectors There are many plasmid vectors available that are compatible with the SuperScript Choice System for cdna Synthesis. Since the SuperScript Choice System is designed to produce cdna containing EcoR I-cohesive ends, the plasmid must contain a unique EcoR I site within the multiple cloning site. When choosing a plasmid consider what screening method will be used and if the library will be used in a subtractive cdna library construction. If the library will be used for making RNA, in vitro translation, or subtraction procedures the multiple cloning site must be flanked by RNA polymerase promoters (i.e. SP6, T7, or T3 RNA polymerase promoters). A vector with an f1 origin of replication can be infected with M13K07 helper phage to produce single-strand plasmid DNA for sequencing or in vitro mutagenesis (22). Nested deletions for sequencing are facilitated by having restriction endonuclease sites in the multiple cloning site clustered together. Plasmid shuttle vectors for transient expression of cloned genes in mammalian cells such as COS cells and DNA cloning in E. coli can also be used with the SuperScript Choice System for cdna Synthesis. Plasmid pcdna3 (+) is a multifunctional vector for cdna cloning, in vitro transcription, and dideoxy sequencing. The plasmid contains a unique multiple cloning site with restriction sites for EcoR I and 11 other restriction endonucleases. There is a T7 RNA polymerase promoter that can be used to generate RNA for probes, in vitro translation, or subtracted cdna libraries. The plasmid contains the CMV promoter, for expression of cloned genes. DNA inserts can be sequenced from double-stranded DNA using T7 forward or BGH reverse sequencing primers. Singlestranded plasmid DNA for sequencing or in vitro mutagenesis can be generated by infection of transformed cells with an appropriate helper phage such as M13K07 (22). The β-lactamase gene on the plasmid provides for convenient selection by ampicillin resistance λ Vectors λgt10, EcoR I Arms is a precut version of λgt10, purified from bacteriophage DNA (imm 434 b527), which has been prepared by ligation at the cos sites, digestion with EcoR I, and dephosphorylation. This vector contains an EcoR I site within the repressor gene and can accommodate inserts up to 7 kb in length (16). Interruption of the repressor gene by insertion of cdna into the EcoR I site converts the phage from cl + to cl -, which changes plaque morphology from turbid to clear when plated on an E. coli strain such as C600 (24). By using the bacterial strain C600hflA150 that contains a high frequency lysogeny mutation (16), lytic growth is repressed so effectively that plaque formation by cl + phage does not occur; only cl - (recombinant) phage produce plaques, significantly reducing the background. Because λgt10 is not an expression vector, recombinant libraries prepared in λgt10 cannot be screened with antibodies. 8

13 λgt11, EcoR I Arms is a precut version of λgt11, purified from bacteriophage DNA (lac5 cl857 nin5 Sam100), which has been prepared by ligation at the cos sites, digestion with EcoR I, and dephosphorylation. Unlike λgt10, λgt11 is an expression vector: libraries prepared in λgt11 can be screened immunologically using specific antibodies (18,25), as well as with nucleic acid probes. This vector contains a unique EcoR I cloning site near the end of a β-galactosidase coding sequence and can accept inserts up to 7.2 kb in length. If cdna containing an open reading frame is inserted into this site in the correct orientation, a fusion protein is produced when expression from the lac promoter is induced with isopropylthio-β-galactoside (IPTG). Thus, expression of proteins, which may be toxic to the cell, can be delayed until the library has been amplified and is ready for immunological screening. The lac promoter is also used to control expression of the cloned gene if large-scale synthesis is needed for purification. Insertion of cdna into the lacz gene of λgt11 will produce plaques that are usually colorless instead of blue on plates containing IPTG and X-gal. The ratio of colorless to blue plaques is often used to estimate the percentage of recombinants in the library. λziplox, EcoR I Arms is a precut version of λziplox, which has been prepared by ligation at the cos sites, digestion with EcoR I, and dephosphorylation. Libraries prepared in λziplox can be screened immunologically using specific antibodies (18,25), as well as with nucleic acid probes. λziplox contains the plasmid pzl1, flanked by loxp sequences, between portions of the left and right arms of λgt10 and λgt11. cdna inserts cloned into the EcoR I site of λziplox reside within the inducible lac Z gene commonly found in puc-type plasmid vectors. When the lac promoter is induced with IPTG, the cloned gene is expressed as a fusion protein embedded within the amino-terminal portion of the β-galactosidase fragment encoded by lac Z. Following cloning and selection of desired clones, the cdna can be recovered in the autonomously replicating multifunctional plasmid pzl1 using a simple in vivo excision protocol, obviating the need for tedious subcloning. Life Technologies vector sequences, restriction information, and maps can be found in the Vector Data area of our web site 2 9

14 Overview 2.9 Ligation of Size-Fractionated cdna to the Vector of Choice The ligation reactions described in Sections 3.8, 3.9 and 3.10 in Chapter 3 will suffice for most applications. We have found that ng of cdna saturates 50 ng of plasmid vector and that 50 ng of cdna saturates 500 ng of λ vector; to use more cdna is wasteful (as little as 1 ng of cdna can be used in either ligation reaction). For any particular population of cdna, however, these ratios may not be optimal. If the described ligation conditions do not yield enough transformants or plaques to make your library complete, the vector-to-cdna ratio yielding the maximum number of clones should be determined empirically. The yield is also dependent upon the transformation efficiency of the E. coli cells used to plate the plasmid-based library or the efficiency of the in vitro packaging methods used to introduce the λ-ligated cdna into E. coli by infection. For either type of vector, the following considerations should be noted: Plasmid Vectors. If the E. coli competent cells yield ~ transformants/µg of puc19 plasmid DNA, then the ligated cdna should yield 0.5 to transformants/µg of vector; this is equivalent to 2.5 to transformants/µg of cdna. Thus, a plasmid library containing clones can be constructed from 10 ng of cdna used in the ligation reaction in Section 3.8. Plasmidligated cdna can also be introduced into E. coli cells by electroporation, which generally will yield a greater number of transformants ( to transformants/µg cdna) from the same amount of ligated cdna. Electroporation may be especially useful if the library must be very large or if you have <10 ng of cdna. λ Vectors. If the in vitro packaging extract yields approximately plaque forming units (pfu)/µg of ligated, wild-type λ DNA, then the ligated cdna should yield to pfu/µg of vector; this is equivalent to to pfu/µg of cdna. Thus, a library containing clones can be constructed from as little as 10 ng of cdna. 10

15 Methods Components The components of the SuperScript Choice System for cdna Synthesis are as follows. Components are provided in sufficient quantities to perform three separate experiments, each converting up to 5 µg of mrna into size-fractionated, EcoR I-adapted cdna, ready for ligation into any EcoR I-digested, dephosphorylated vector. Store chromatography columns (Part No. 8092CL) at 2 C to 8 C and store the reagent assembly (Part No. 8090RT) at 30 C to 10 C. Component Amount Oligo(dT) primer (0.5 µg/µl) µl Random Hexamers (50 ng/µl) µl 5X First-Strand Buffer [250 mm Tris-HCl (ph 8.3), 375 mm KCl, 15 mm MgCl 2 ]... 1 ml 0.1 M DTT µl 10 mm dntp Mix (10 mm each datp, dctp, dgtp, dttp) µl SuperScript II RT (200 units/µl) µl 5X Second-Strand Buffer [100 mm Tris-HCl (ph 6.9), 450 mm KCl, 23 mm MgCl 2, 0.75 mm ß-NAD +, 50 mm (NH 4 ) 2 SO 4 ] µl E. coli DNA Ligase (10 units/µl) µl E. coli DNA Polymerase I (10 units/µl) µl E. coli RNase H (2 units/µl) µl T4 DNA Polymerase (5 units/µl) µl 5X Adapter Buffer [330 mm Tris-HCl (ph 7.6), 50 mm MgCl 2, 5 mm ATP] µl EcoR I (Not I) Adapters (1 µg/µl) µl T4 DNA Ligase (1 units/µl) µl T4 Polynucleotide Kinase (10 units/µl) µl 5X T4 DNA Ligase Buffer [250 mm Tris-HCl (ph 7.6), 50 mm MgCl 2, 5 mm ATP, 5 mm DTT, 25% (w/v) PEG 8000]... 1 ml DEPC-treated Water ml Control RNA (0.5 µg/µl) µl Yeast trna (1 µg/µl) µl cdna size fractionation columns... three Manual... one 3.2 General Comments mrna Purification One of the most important steps preceding the synthesis of cdna and the establishment of a library is isolation of intact mrna. The Micro-FastTrack 2.0 mrna Isolation Kit allows you to isolate mrna in a fast and convenient procedure. Successful cdna synthesis demands an RNase-free environment at all times, which will generally require the same level of care used to maintain aseptic condi tions when working with microorganisms. Several additional guidelines should be followed: 1. Never assume that anything is RNase-free, except sterile pipets, centrifuge tubes, culture tubes, and any similar labware that is explicitly stated to be sterile. 2. Dedicate a separate set of automatic pipets for manipulating RNA and the 11

16 Methods Note: The use of pre-made phenol: chloroform:isoamyl alcohol (25:24:1, v/v) is recommended. If making your own, saturate the redistilled phenol with TEN buffer, not with distilled water. Note: Suggested stopping points are noted in the protocol with the icon. buffers and enzymes used to synthesize cdna. All purpose pipets may be quickly prepared for RNA use by wiping the outside surface of the pipet with RNase AWAY Reagent. Use barrier tips such as ART Tips. 3. Obtain RNase-free microcentrifuge tubes or treat tubes overnight in a 0.01% (v/v) aqueous solution of diethylpyrocarbonate (DEPC); rinse them with autoclaved, distilled water; and autoclave them. 4. Avoid using any recycled glassware unless it has been specifically rendered RNase-free by rinsing with 0.5 N NaOH or RNase AWAY Reagent followed by copious amounts of DEPC-treated, autoclaved water or other prepared RNase-free water. Alternatively, bake glassware at 150 C for 4 hours. 5. Use reagent grade solutions that are set aside for RNA use only. The preferred method of obtaining RNase-free solutions is to treat them with 0.01% (v/v) DEPC followed by autoclaving. If preparing solutions containing primary amines (such as Tris), where DEPC cannot be used, or preparing heatsensitive solutions, use RNase-free reagents, water and labware, and filter the solution through a 0.2-µm disposable, sterile filter Advance Preparations Before using this system, please review the protocol flow diagram in figure 4. You will need the following items not included in this system: autoclaved 1.5-mL microcentrifuge tubes microcentrifuge capable of generating a relative centrifugal force of 14,000 g automatic pipets capable of dispensing 1 to 20 µl, 20 to 200 µl, and 200 µl to 1 ml autoclaved, disposable tips for automatic pipets disposable gloves 16 C and 37 C water baths 1 10 µci [α- 32 P]dCTP (400 to 3,000 Ci/mmol) 500 ml 10% (w/v) TCA (trichloroacetic acid) containing 1% (w/v) sodium pyrophosphate (store at 4 C) glass fiber filters (1 2 cm) (Whatman GF/C or equivalent) buffer-saturated phenol:chcl 3 :isoamyl alcohol [25:24:1 (v/v/v)] TEN buffer [10 mm Tris-HCl (ph 7.5), 0.1 mm EDTA, 25 mm NaCl; autoclaved] 7.5 M ammonium acetate (NH 4 OAc) filtered through a sterile, 0.2-µm nitrocellulose filter 70% (v/v) ethanol ( 20 C) Time Planning Starting with poly(a) + RNA, these protocols are designed to yield cdna containing EcoR I-cohesive ends in ~2 days. For best results, the procedure should be completed as quickly as possible because radiochemical effects induced by the decay of the 32 P in the cdna can diminish transformation efficiencies over time. We recommend that the protocols be completed as follows: Day 1: Sections 3.3 and 3.4, and steps 1 and 2 of Section 3.5 (overnight incubation of EcoR I (Not I) Adapter Addition reaction). Day 2: Sections 3.5 through 3.8 or 3.9. If interrupting the procedure at any other point becomes necessary, you may do so following Sections 3.4 or 3.7. When stopping at any such point, always store the cdna as the uncentrifuged ethanol precipitate at 20 C to minimize the aforementioned effects of 32 P decay Utilization of Reagents Reagents included with the SuperScript Choice System are provided in sufficient quantities to perform three complete experiments converting up to 5 µg of mrna into size-fractionated, EcoR I-adapted cdna. Additionally, the components for first 12

17 3 Poly(A) + mrna 70 C, 10 min 37 C, 2 min Oligo(dT) 12-18, Random Hexamers,or both DEPC-Treated Water 5X First-Strand Buffer4 0.1 M DTT 10 mm dntp Mix [α- 32 P]dCTP SuperScript II RT First-strand reaction Remove aliquot for yield and gel electrophoretic analyses 37 C, 1 h First-strand cdna Transfer to ice DEPC-treated water 5X Second-Strand Buffer 10 mm dntp Mix E. coli DNA Ligase E. coli DNA Polymerase I E. coli RNase H Second-strand reaction 16 C, 2 h 16 C, 5 min T4 DNA Polymerase Extract, precipitate ds cdna 16 C, 16 h 70 C, 10 min DEPC-Treated Water 5X Adapter Buffer EcoR I (Not I) Adapters 0.1 M DTT T4 DNA Ligase EcoR I-adapted cdna 37 C, 30 min 70 C, 10 min T4 Polynucleotide Kinase Column chromatography Size-fractionated cdna Figure 4. Detailed protocol flow diagram. 13

18 Methods Note: The efficiency of the second-strand reaction is influenced by the amount and concentration of the reactants, so the instructions must be followed as described for best results. Note: Do not proceed with this protocol until you have made the appropriate decisions regarding your choice of primer. For more information, see Section 2.3, Choosing the Priming Method. and second-strand synthesis are provided in sufficient quantities to perform Sections 3.3 and 3.4 five times. You may wish to use these extra quantities to test a small amount of your mrna by determining first or second-strand yield and visualizing the distribution of the products by gel electrophoresis. Alternatively, the extra components can be held as a backup in case of accidental loss of material or procedural error. 3.3 First-Strand Synthesis The 20-µL reaction described is designed to convert up to 5 µg of mrna into firststrand cdna. The amount of SuperScript II RT added to the reaction will be dependent upon the amount of starting mrna. We recommend 200 units of SuperScript II RT for 1 µg of mrna and 200 units/µg of mrna for 1 5 µg of mrna. If second-strand cdna is to be labeled instead of first-strand cdna, the first-strand reaction should be set up without [α- 32 P]dCTP (adjust the amount of water in the reaction to maintain the 20-µL final volume), and the reaction should be carried through the second-strand synthesis procedure as described in Section 3.4. In this case, add 1 µl (10 µci/µl) [α- 32 P]dCTP to the second-strand reaction after the 10 mm dntp mix is added. A single-transcript control RNA is included in the SuperScript Choice System as an aid in verifying the first-strand reaction. If you decide to use the control RNA, simply substitute 4 µl (2 µg) in the first-strand reaction for your mrna. 1. Perform one of the following substeps a, b, or c depending on your choice of priming method. a. For priming with oligo(dt): Add 2 µl of Oligo(dT) primer to a sterile 1.5-mL microcentrifuge tube. Add mrna, diluted as needed with DEPCtreated water, according to the following table: µg of mrna mrna (plus DEPC-treated water) to a total volume (µl) b. For priming with random hexamers: Add 1 to 3 µl (50 to 150 ng) of Random Hexamers to a sterile 1.5-mL microcentrifuge tube. Add mrna, diluted as needed with DEPC-treated water, according to the following table: mrna (plus DEPC-treated water) µg of mrna to a total volume (µl) c. For priming with both Oligo(dT) and Random Hexamers: Add 2 µl of Oligo(dT) Primer and 1 to 3 µl (50 to 150 ng) of Random Hexamers to a sterile 1.5-mL microcentrifuge tube. Add mrna, diluted as needed with DEPC-treated water, according to the following table: mrna (plus DEPC-treated water) µg of mrna to a total volume (µl)

19 2. Heat the mixture to 70 C for 10 minutes and quick-chill on ice. Collect the contents of the tube by brief centrifugation and add the following: Component Volume (µl) 5X First-Strand Buffer M DTT 2 10 mm dntp Mix 1 [α- 32 P]dCTP (1 µci/µl) 1 The total volume should now correspond to the following table: µg of mrna (from step 1) Total volume (µl) Mix the contents of the tube by gently vortexing and collect the reaction by brief centrifugation. Place the tube at 37 C for 2 minutes to equilibrate the temperature. 4. Add SuperScript II RT according to the following table: µg of mrna (from step 1) SuperScript II RT (µl) Mix gently and incubate at 37 C for 1 hour. Regardless of the amount of starting mrna, the total volume should now be 20 µl. Final composition of the reaction: 50 mm Tris-HCl (ph 8.3) 75 mm KCl 3 mm MgCl 2 10 mm DTT 500 µm each datp, dctp, dgtp, and dttp 50 µg/ml oligo(dt) primer and/or µg/ml random hexamers 5 µg ( 250 µg/ml) mrna 10,000 50,000 units/ml SuperScript II RT 5. Place the tube on ice to terminate the reaction. 6. Remove 2 µl from the reaction and add it to a microcentrifuge tube containing 43 µl of 20 mm EDTA (ph 7.5) and 5 µl of Yeast trna. This mixture will be used in calculating first-strand yield. 7. Take the remaining 18 µl of the first-strand reaction and continue immediately with the first two steps of the second-strand reaction as described in Section While the second-strand reaction is incubating, spot duplicate 10-µL aliquots from the diluted sample from step 6 of this section onto glass fiber filters. Dry one of the filters under a heat lamp or at room temperature. This filter will be used to determine the specific activity of the dctp reaction. 9. Wash the other filter three times in sequence, for 5 minutes each time, in a beaker containing 50 ml of fresh, ice-cold 10% (w/v) TCA containing 1% (w/v) sodium pyrophosphate. Wash the filter once with 50 ml of 95% ethanol at room temperature for 2 minutes. Dry the filter under a heat lamp or at room temperature. This filter will be used to determine the yield of first-strand cdna. 10. Count both filters in standard scintillant to determine the amount of 32 P in the reaction, as well as the amount of 32 P that was incorporated. See Section 3.11, Analysis of cdna Products, for information needed to convert the data into yield of first-strand cdna. 15

20 Caution: If the first-strand cdna was labeled, the supernatant(s) will be radioactive. Dispose of this material properly. Caution: If the first or second-strand cdna was labeled, the supernatant(s) will be radioactive. Dispose of this material properly. 11. Precipitate the remaining 30 µl of the sample from step 6 of this section by adding 15 µl of 7.5 M NH 4 OAc, followed by 90 µl of absolute ethanol ( 20 C). Vortex the mixture thoroughly and immediately centrifuge at room temperature for 20 minutes at 14,000 g. 12. Remove the supernatant carefully, and overlay the pellet with 0.5 ml of 70% ethanol ( 20 C). Centrifuge for 2 minutes at 14,000 g and remove the supernatant. 13. Dry the cdna at 37 C for 10 minutes to evaporate residual ethanol and proceed to Section 3.11, Analysis of cdna Products. 3.4 Second-Strand Synthesis This protocol is suitable for synthesizing second-strand cdna from 5 µg mrna originally in the 20-µL first-strand reaction. 1. On ice, add the following reagents, in the order shown, to the first-strand reaction tube: Component Volume (µl) DEPC-Treated Water X Second-Strand Buffer mm dntp Mix...3 E. coli DNA Ligase (10 units/µl)...1 E. coli DNA Polymerase I (10 units/µl)...4 E. coli RNase H (2 units/µl)...1 Final volume Final composition of the reaction: 25 mm Tris-HCl (ph 7.5) 100 mm KCl 5 mm MgCl 2 10 mm (NH 4 ) 2 SO mm ß-NAD µm each datp, dctp, dgtp, dttp 1.2 mm DTT 65 units/ml DNA ligase 250 units/ml DNA polymerase I 13 units/ml RNase H 2. Vortex the tube gently to mix and incubate the completed reaction for 2 hours at 16 C. Do not let the temperature rise above 16 C. 3. Add 2 µl (10 units) of T4 DNA Polymerase and continue incubating at 16 C for 5 minutes. 4. Place the reaction on ice and add 10 µl of 0.5 M EDTA. Note: If [α- 32 P]dCTP was added to the second-strand reaction, remove 10 µl from the reaction, and add it to a microcentrifuge tube containing 35 µl of 20 mm EDTA (ph 7.5) and 5 µl of Yeast trna. This mixture will be used in calculating second-strand yield. Then proceed as described in steps 8 to 10 in Section 3.3 for processing and counting the filters. 5. Add 150 µl of phenol:chloroform:isoamyl alcohol (25:24:1), vortex thoroughly, and centrifuge at room temperature for 5 minutes at 14,000 g to separate the phases. Carefully remove 140 µl of the upper, aqueous layer, and transfer it to a fresh 1.5-mL microcentrifuge tube. 6. Add 70 µl of 7.5 M NH 4 OAc, followed by 0.5 ml of absolute ethanol ( 20 C). Vortex the mixture thoroughly and immediately centrifuge at room temperature for 20 minutes at 14,000 g. 7. Remove the supernatant carefully and overlay the pellet with 0.5 ml of 70% ethanol ( 20 C). Centrifuge for 2 minutes at 14,000 g and remove the supernatant. 8. Dry the cdna at 37 C for 10 minutes to evaporate residual ethanol and proceed to Section

21 Note: Do not add more than one reaction per column. Addition of multiple reactions will result in poor cloning efficiency. Note: If the flow rate is noticeably slower than 20 min/ml, do not use the column. Additionally, if the drop size from the column is not ~25 to 35 µl, do not use the column. The integrity and resolution of the cdna might be compromised. Tip: When collecting fractions, wear gloves that have been rinsed with ethanol to reduce static. Also, position the tube 1 to 2 cm from the bottom of the column to avoid the effects of static on drop size. 3.5 EcoRI (Not I) Adapter Addition 1. Add the following reagents on ice, in the order shown, to the cdna from step 8 of Section 3.4. Component Volume (µl) DEPC-treated Water X Adapter Buffer...10 EcoR I (Not I) Adapters M DTT...7 T4 DNA Ligase...5 Final volume...50 Final composition of the reaction: 66 mm Tris-HCl (ph 7.6) 10 mm MgCl 2 1 mm ATP 14 mm DTT 200 µg/ml EcoR I (Not I) adapters 100 units/ml T4 DNA ligase 2. Mix gently and incubate the reaction at 16 C for a minimum of 16 hours. For greatest convenience, simply let the reaction proceed overnight. 3. Heat the reaction at 70 C for 10 minutes to inactivate the ligase. 4. Place the reaction on ice and proceed to Section Phosphorylation of EcoR I-Adapted cdna 1. Add 3 µl of T4 Polynucleotide Kinase to the reaction from step 4 of Section Mix gently and incubate the reaction for 30 minutes at 37 C. Note: While the reaction is incubating, you may begin performing steps 1 through 3 of Section Heat the reaction at 70 C for 10 minutes to inactivate the kinase. 4. Place the reaction on ice and proceed to Section Column Chromatography This procedure optimizes size fractionation of the cdna and makes the cloning of larger inserts more probable. This procedure also ensures that residual adapters do not enter into the library. Failure to adhere to these instructions can compromise the quality of your cdna library. 1. Place one of the cdna size fractionation columns in a support. Remove the top cap first, and then the bottom cap. Allow the excess liquid (20% ethanol) to drain. 2. Pipet 0.8 ml of TEN buffer [10 mm Tris-HCl (ph 7.5), 0.1 mm EDTA, 25 mm NaCl; autoclaved] onto the upper frit and let it drain completely. Repeat this step three more times for a total of 3.2 ml. Each 0.8-mL wash will take approximately 15 minutes, but it is important to do all four washes to remove the 20% ethanol from the column. 3. Label 20 sterile microcentrifuge tubes from 1 to 20, and place them in a rack with tube 1 under the outlet of the column. 4. Add 97 µl of TEN buffer to the cdna reaction from step 4 of Section 3.6 and mix gently. 5. Add the entire sample to the center of the top frit and let it drain into the bed. Collect the effluent into tube Add 100 µl of TEN buffer to the column and collect the effluent into tube 2. Note: Let the column drain completely in other words, until it stops dripping before the addition of each new 100-µL aliquot. 7. Beginning with the next 100-µL aliquot of TEN buffer, collect single-drop (~35 µl) fractions into individual tubes. Continue adding 100-µL aliquots of TEN buffer until you have collected a total of 18 drops into tubes 3 through 20, one drop per tube. 3 17

22 Methods 8. Using an automatic pipet, measure the volume in each tube; use a fresh tip for each fraction to avoid cross-contamination. Record each value in column A of one of the following work tables, using the sample table as a guide. Cap each tube after the volume has been measured and recorded. Calculate the cumulative elution volume with the addition of each fraction and record this value in column B. Note: The sample table contains data typical for the size fractionation protocol when oligo(dt) has been used to prime first-strand cdna synthesis; actual data will vary. The sample is provided merely to illustrate the decision-making process for selecting cdna for the vector ligation reaction. 9. Identify the fraction for which the value in column B is closest to, but not exceeding, 600 µl (corresponding to fraction 12 in the sample table). Draw a horizontal line across the table immediately below this fraction. Do not use any of the subsequent fractions for your cdna library; remove them to a separate tube rack to avoid accidentally using them in the remainder of the protocol. Important: Fractions collected after the 600-µL cutoff point (corresponding to tubes 13 through 20 in the sample table) will contain smaller cdnas and unligated adapters. Use of these fractions significantly increases the risk of cloning the EcoR I (Not I) adapters. In some cases with random hexamerprimed reactions, the target cdna may elute in a later fraction, in which case taking fractions beyond the passage of 600 µl may be a necessary risk. 10. Place the remaining tubes in a scintillation counter and obtain Cerenkov counts for each fraction. Count the entire sample in the tritium channel; do not add scintillation fluid to the tubes. In column C, record the counts corresponding to each fraction. Note: Cerenkov counts above background should appear after passage of µl of buffer. 11. For each fraction in which the Cerenkov counts exceed background (corresponding to fractions 8 to 12 in the sample table), calculate the amount of cdna, using equation 5 in Section 3.12, Analysis of cdna from the cdna Size Fractionation Column. Record each cdna amount in column D. 12. Divide each cdna amount in column D by the fraction volume given in column A to determine the cdna concentration per fraction. Record this value in column E. 13. The plasmid vector ligation reaction in Section 3.8 requires 10 ng of cdna at 1 ng/µl. The λ vector ligation in Section 3.9 requires 50 ng of cdna as a dried pellet. Examine the data entered in columns D and E of your work table and decide which fractions to pool and precipitate early fractions, or (if applicable), to use the appropriate amount of cdna from a suitable fraction directly in the selected vector ligation reaction. Guidelines for making this decision are provided in Section 3.12, Analysis of cdna from the cdna Size Fractionation Column. 14. If cdna from two or more fractions must be pooled to obtain the cdna needed for the vector ligation reaction, uncap the first selected tube (corresponding to fraction 8 in the sample table), and add cdna from each subsequent fraction until there is the correct amount of cdna (as determined in step 13) in the tube. Measure the volume and add 5 µl of Yeast trna to the tube. 15. Add 0.5 volumes of NH 4 OAc, followed by 2 volumes of absolute ethanol ( 20 C). Vortex the mixture thoroughly and immediately centrifuge at room temperature for 20 minutes at 14,000 g. Note: If you are stopping here (see Time Planning under Section 3.2, General Comments), store the tubes at 20 C overnight before centrifugation to minimize the effects of 32 P decay. 16. Remove the supernatant carefully and overlay the pellet with 0.5 ml of 70% ethanol ( 20 C). Centrifuge for 2 minutes at 14,000 g and remove the supernatant. 17. Dry the cdna at 37 C for 10 minutes to evaporate residual ethanol. To ligate to a plasmid vector, dissolve the cdna in 10 µl of TEN buffer and proceed to Section 3.8. For ligation to a λ vector, proceed directly to Section

23 3 Sample Experiment Experiment 1 No. A Fraction Volume (µl) B Total Volume (µl) C Cerenkov Counts (CPM) D Amount of cdna (ng) E Concentration of cdna (ng/µl) No. A Fraction Volume (µl) B Total Volume (µl) C Cerenkov Counts (CPM) D Amount of cdna (ng) E Concentration of cdna (ng/µl) , , ,

24 20 Methods No. A Fraction Volume (µl) B Total Volume (µl) C Cerenkov Counts (CPM) D Amount of cdna (ng) E Concentration of cdna (ng/µl) Experiment 2 No. A Fraction Volume (µl) B Total Volume (µl) C Cerenkov Counts (CPM) D Amount of cdna (ng) E Concentration of cdna (ng/µl) Experiment

25 Note: Although 10 to 20 ng of cdna generally saturates the vector, the amount of cdna that yields the maximal number of clones may be higher (e.g., 40 ng). As much as 14 µl of cdna in TEN buffer may be added to the ligation reaction. Note: Although 20 to 50 ng of cdna generally saturates the vector, the amount of cdna can be varied to determine what quantity yields the maximal number of clones. 3.8 Ligation of cdna to a Plasmid Vector If you intend to ligate the cdna to a λ vector, use Section 3.9 or This protocol is intended for use with 10 ng of cdna. Note: Do not proceed with this protocol until you have made the appropriate decisions regarding the choice of fractions for use in the ligation reaction. For more information, refer to Section 3.12, Analysis of cdna from the cdna Size Fractionation Column. 1. Add the following, at room temperature, to a sterile 1.5-mL microcentrifuge tube: Component Amount 5X T4 DNA Ligase Buffer...4 µl plasmid vector, EcoR I-cut, dephosphorylated (50 ng/µl)...50 ng cdna ( 1 ng/µl)...10 ng DEPC-Treated Water...sufficient to bring the volume to 19 µl 2. Add 1 µl of T4 DNA Ligase and mix by pipetting. Final composition of the reaction: 50 mm Tris-HCl (ph 7.6) 10 mm MgCl 2 1 mm ATP 5% (w/v) PEG mm DTT 2.5 µg/ml plasmid vector, EcoR I-Cut 0.5 µg/ml cdna 50 units/ml T4 DNA ligase 3. Let the reaction incubate for 3 hours at room temperature or overnight at 4 C. Note: Following incubation, the cdna will be ligated into the cloning vector and ready for transformation into E.coli cells such as MAX Efficiency DH5α or DH10B Competent Cells, or for precipitation procedures prior to electroporation into cells such as ElectroMAX DH10B Cells. See Section 6 for information on these products. 3.9 Ligation of cdna to λgt11 and λgt10 Vectors If you intend to ligate the cdna to a plasmid vector, use Section 3.8. This protocol is intended for use with 50 ng of cdna as a dried pellet. Note: Do not proceed with this protocol until you have made the appropriate decisions regarding the choice of fractions for use in the ligation reaction. For more information, refer to Section 3.12, Analysis of cdna from the cdna Size Fractionation Column. 1. Add the following, at room temperature, to a sterile 1.5-mL microcentrifuge tube containing the dried cdna pellet: Component Amount 5X T4 DNA Ligase Buffer...1 µl λ vector, EcoR I Arms (250 ng/µl) ng cdna (as a dried pellet)...50 ng DEPC-treated water... sufficient to bring the volume to 4 µl Mix by pipetting to ensure that the cdna is completely dissolved. 2. Add 1 µl of T4 DNA Ligase and mix by pipetting. Final composition of the reaction: 50 mm Tris-HCl (ph 7.6) 10 mm MgCl 2 1 mm ATP 5% (w/v) PEG mm DTT 100 µg/ml λ vector, EcoR I Arms 10 µg/ml cdna 200 units/ml T4 DNA ligase 3 21

26 Methods Note: Although 20 to 50 ng of cdna generally saturates the vector, the amount of cdna can be varied to determine what quantity yields the maximal number of clones. 3. Let the reaction incubate for 3 hours at room temperature or overnight at 4 C. Note: Following incubation, the cdna will be ligated into the cloning vector and ready for in vitro packaging Ligation of cdna to a λziplox Vector 1. Prepare a 5X DNA ligase buffer separately. Do not use the 5X T4 DNA Ligase Buffer supplied with this system; it contains PEG which inhibits the packaging of λziplox. Mix 10 µl of the 10X DNA Ligase Buffer, supplied with the λziplox [400 mm Tris-HCl (ph 7.5), 100 mm MgCl 2, 15 mm ATP] with 10 µl of 100 mm DTT in a microcentrifuge tube on ice. 2. Add the following, at room temperature, to a sterile 1.5-mL microcentrifuge tube containing the dried cdna pellet: Component Amount 5X DNA Ligase buffer (from step 1)...1 µl λziplox Arms, Not I - Sal I (250 ng/µl)... 2 µl cdna (as a dried pellet)...20 to 50 ng distilled water...1 µl Mix by pipetting to ensure that the cdna is completely dissolved. 3. Add 1 µl (one unit) of T4 DNA ligase and mix gently by pipetting. 4. Let the reaction incubate for 3 hours at room temperature or overnight at 4 C. 5. Following incubation, the cdna will be ligated into the cloning vector and ready for in vitro packaging Analysis of cdna Products First-Strand Yield The overall yield of the first-strand reaction is calculated from the amount of acidprecipitable radioactivity determined as described in Section 3.3. In order to perform the calculation, you must first determine the specific activity (SA) of the radioisotope in the reaction. The specific activity is defined as the counts per minute (cpm) of an aliquot of the reaction divided by the quantity (in pmol) of the same nucleotide in the aliquot. For [α- 32 P]dCTP, the specific activity is given by the relationship: cpm/10 µl SA (cpm/pmol dctp) = [1] 200 pmol dctp/10 µl The amount of dctp contributed by the radiolabeled material is insignificant relative to the unlabeled nucleotide and is ignored in equation 1. Once the specific activity is known, the amount of cdna in the first-strand reaction can be calculated from the amount of acid-precipitable radioactivity determined from the washed filter: Amount of ds cdna (µg) (cpm) (50 µl/10 µl) (20 µl/2 µl) (4 pmol dntp/pmol dctp) = [2] (cpm/pmol dctp) (3,030 pmol dntp/µg cdna) The correction in the numerator takes into account that, on the average, four nucleotides will be incorporated into the cdna for every dctp scored by this assay. The factor in the denominator is the amount of nucleotide that corresponds to 1 µg of single-stranded DNA. Example: The unwashed filter gave 50,000 cpm when it was counted. The specific activity of the dctp is given by equation 1: SA (cpm/pmol dctp) = 50,000 cpm/10 µl 200 pmol dctp/10 µl = 250 cpm/pmol dctp 22

27 If 2 µg of starting mrna was used and the washed filter gave 1,800 cpm, then the amount of cdna is calculated using equation 2: Amount of (1,800 cpm) (50 µl/10 µl) (20 µl/2 µl) (4 pmol dntp/pmol dctp) = ds cdna (µg) (250 cpm/pmol dctp) (3,030 pmol dntp/µg cdna) = 0.5 µg first-strand cdna This amount of first-strand cdna would represent a 25% yield relative to the 2 µg of mrna starting material Second-Strand Yield The overall yield of the second-strand reaction is calculated from the amount of acid-precipitable radioactivity determined as described in Section 3.4. In order to perform the calculation, you must first determine the specific activity of the radioisotope in the reaction. The specific activity is defined as the counts per minute of an aliquot of the reaction divided by the quantity of the same nucleotide in the aliquot. For [α- 32 P]dCTP, the specific activity is given by the relationship: cpm/10 µl SA (cpm/pmol dctp)= [3] 500 pmol dctp/10 µl 3 The amount of dctp contributed by the radiolabeled material is insignificant relative to the unlabeled nucleotide and is ignored in equation 3. Once the specific activity is known, the amount of cdna in the second-strand reaction can be calculated from the amount of acid-precipitable radioactivity determined from the washed filter: Amount of cdna (µg) (cpm) (50 µl/10 µl) (150 µl/10 µl) (4 pmol dntp/pmol dctp) = [4] (cpm/pmol dctp) (3,030 pmol dntp/µg cdna) The correction in the numerator takes into account that, on the average, four nucleotides will be incorporated into the cdna for every dctp scored by this assay. The factor in the denominator is the amount of nucleotide that corresponds to 1 µg of single-stranded DNA. Example: The unwashed filter gave 300,000 cpm when it was counted. The specific activity of the dctp is given by equation 3: 300,000 cpm/10 µl SA (cpm/pmol dctp)= 500 pmol dctp/10 µl = 600 cpm/pmol dctp If 2 µg of starting mrna was used and the washed filter gave 2,500 cpm, then the amount of cdna is calculated using equation 4: Amount of dctp) cdna (µg) = (2,500 cpm) (50 µl/10 µl) (150 µl/10 µl) (4 pmol dntp/pmol (600 cpm/pmol dctp) (3,030 pmol dntp/µg cdna) Gel Analysis = 0.4 µg second-strand cdna The first or second-strand cdna, if labeled with 32 P, can be analyzed by alkaline agarose gel electrophoresis to estimate the size range of products synthesized (15,28). The ethanol-precipitated first-strand sample is dissolved in 10 µl 1X alkaline agarose gel sample buffer [30 mm NaOH, 1 mm EDTA, 10% (v/v) glycerol, 0.01% bromophenol blue]. Other samples (such as the 1 Kb DNA Ladder, labeled with 32 P) can be electrophoresed after addition of a suitable volume of a more concentrated sample buffer; the only precaution is to chelate any Mg 2+ by addition of EDTA prior to adding the alkaline sample buffer. 23

28 Methods The gel [1.4% (w/v)] should be cast in the appropriate volume of 30 mm NaCl, 2 mmedta (alkaline buffer cannot be used because it will degrade the agarose when the solution is microwaved to melt the agarose) and should be equilibrated for 2 to 3 hours in alkaline electrophoresis buffer (30 mm NaOH, 2 mm EDTA) before loading the samples. Electrophoresis should be for 5 to 6 hours at 50 V or for 16 to 18 hours at 15 V. The gel should be dehydrated under vacuum until the buffer is removed, then under heat and vacuum for several hours to complete the drying. The dried gel should then be exposed to x-ray film overnight at room temperature. kb Marker First-Strand Second-Strand When a heterogeneous mrna population is fractionated by alkaline gel electrophoresis, a continuum of fragments ranging in size from 500 to 5,000 nucleotides makes up the bulk of the first and second-strand cdna. Figure 5 shows an alkaline electrophoretic analysis of 32 P-labeled first and second-strand cdna synthesized from oligo(dt)-primed HeLa mrna with the SuperScript Choice System Analysis of cdna from the cdna Size Fractionation Column Calculation of the amount of size-fractionated cdna in each column fraction is necessary to ensure that the proper decisions are made concerning fraction selection and that the cdna is used economically in the ligation reaction. The Cerenkov counts are approximately 50% of the radioactivity that would be measured in scintillant. The counts are converted into nanograms of cdna using the specific activity determined in Section 3.3 or 3.4. The amount of cdna (as double strand) in each fraction is given by the following relationship: Amount of (Cerenkov cpm) 2 (4 pmol dntp/pmol dctp) (1,000 ng/µg ds cdna) = [5] ds cdna (ng) SA (cpm/pmol dctp) (1,515 pmol dntp/µg ds cdna) Example: If one of the fractions from the column gave 1,500 cpm when counted by Cerenkov radiation (first-strand-labeled), then the amount of cdna in that fraction is calculated using equation 5: 1.0 Amount of = ds cdna (ng) (1,500 cpm) 2 (4 pmol dntp/pmol dctp) (1,000 ng/µg ds cdna) SA (cpm/pmol dctp) (1,515 pmol dntp/µg ds cdna) = 32 ng 0.5 Figure 5. Alkaline agarose gel analysis of first and second-strand cdna synthesized with the SuperScript Choice System. Samples of 32 P-labeled first or second-strand cdna made from HeLa mrna were ethanol-precipitated, dissolved in alkaline agarose sample buffer, and electrophoresed on a 1.4% agarose gel at 15 V for 16 h. After calculating the amount and the concentration of cdna in each fraction (columns D and E of the work table), you are ready to select and recover cdna for use in the vector ligation reaction in Section 3.8, 3.9, or Depending on which type of vector you intend to use, certain considerations should be noted: Recovering cdna for ligation to plasmid vectors. If you wish to maximize the average insert size in a plasmid-based cdna library and your earliest selected fraction (corresponding to fraction 8 in the sample table) contains less than the 10 ng of cdna required for Section 3.8, you will need to pool cdna from this fraction with at least a portion of subsequent fractions. Because the resulting cdna solution will be too dilute for use in the ligation reaction, you will also need to ethanolprecipitate the pooled cdna. For example, fraction 8 from the sample table in Section 3.7 contains 3.3 ng of cdna in 34 µl, and fraction 9 contains 16 ng of cdna in 36 µl. If all of fraction 8 is combined with 15 µl from fraction 9, the pool will contain 10 ng of cdna in 49 µl. The ethanol precipitation steps in Section 3.7 will then concentrate the 10 ng cdna in preparation for the plasmid vector ligation reaction in Section 3.8. If any of the selected fractions contain 10 ng of cdna at 1 ng/µl, you can use 10 ng from the fraction directly in the plasmid vector ligation reaction in Section 3.8. Note: If this fraction is not the earliest selected, based on Cerenkov counts (corresponding to fraction 8 in the sample table), the average insert size in your cdna library will be smaller than could be obtained through additional pooling and 24

29 ethanol precipitation steps. Figure 6 provides an electrophoretic display of the size ranges of cdna obtained in fractions 8 through 19 in the experiment that generated the sample table data. Recovering cdna for ligation to λ vectors. If you wish to maximize the average insert size in a λ-based cdna library and your earliest selected fraction (corresponding to fraction 8 in the sample table) contains less than the 50 ng of cdna required for Section 3.9 or 3.10, you will need to pool cdna from this fraction with at least a portion of subsequent fractions. Because the cdna for use in Section 3.9 or 3.10 must be in the form of a dry pellet, you will need to ethanol-precipitate the cdna, pooled or not. For example, pooling all of fractions 8, 9, and 10 from the sample table yields ~50 ng of cdna in solution; the ethanol precipitation steps in Section 3.7 will then concentrate the cdna, and 50 ng from the sample can be used in the λ vector ligation reaction in Section 3.9 or The preceding discussion assumes that you wish to achieve maximum transformation efficiencies in keeping with the design of Section 3.8, 3.9, or One other procedural option is available: if you wish merely to maximize insert size and are willing to accept lower transformation efficiencies to attain this goal, you may use your earliest selected fraction in Section 3.8, 3.9, or 3.10 even if it contains less than the required amount of cdna. Please note that the above option should preferably be attempted in plasmid vectors using electroporation methods, or in λ vectors using in vitro packaging methods, since these methods offer higher cloning efficiencies (and thus a greater chance of generating a sufficiently large cdna library). 3 kb Column Fraction Figure 6. Electrophoretic analysis of size-fractionated cdna. [ 32 P]cDNA was fractionated on a 1-mL prepacked column equilibrated in TEN buffer. Single-drop fractions (~35 µl each) were collected, and aliquots were analyzed by electrophoresis on a 1% agarose gel in 40 mm Tris-acetate (ph 8.3), 5 mm sodium acetate, 1 mm EDTA. The gel was electrophoresed at 200 V for 2 hours. 25