Copy Kit. Version G Copy Kit. cdna Synthesis System. Catalog no. L

Transcription

1 Copy Kit Version G Copy Kit cdna Synthesis System Catalog no. L tech_service@invitrogen.com

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3 Table of Contents Table of Contents...iii Kit Contents and Storage... v Accessory Products...vii Introduction... 1 Overview... 1 Experimental Overview... 3 Methods... 4 Isolating RNA... 4 cdna Synthesis... 5 Appendix... 9 Determining the Yield of First-Strand Synthesis... 9 Procedure Checklist for cdna Synthesis Technical Service Product Qualification References iii

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5 Kit Contents and Storage Kit Contents and Storage The Copy Kit contains two modules, the cdna Synthesis Module and the Enzyme Module. The components included in the two modules are described below. Store both modules at -80 C until use. Once the kit has been used, store the components at -20 C. Minimize freezing and thawing of the reagents. The Copy Kit includes sufficient reagents to perform five cdna synthesis reactions. The caps of the vials in the kit are color coded to identify reagents that are used together. cdna Synthesis Module The components of the cdna Synthesis Module are described in the table below. Store at -20 C. Vial Reagent Composition Quantity RT1 Sterile Water Sterile, DEPC-treated water 1.5 ml RT2 5X Reverse Transcriptase (RT) Buffer 500 mm Tris-HCl, ph mm KCl 75 µl 50 mm MgCl mm spermidine RT3 100 mm dntp 100 mm dgtp 100 mm datp 100 mm dctp 100 mm dttp in DEPC water 14 µl RT4 Sodium Pyrophosphate 80 mm in DEPC water 25 µl RT5 Oligo dt (Not I) Primer 0.2 µg/ µl in TE buffer 30 µl RT6 Random Primer (N6) 1.0 µg/ µl in DEPC water 30 µl S1 1X Second Strand Buffer 191 mm KCl 1.0 ml 4.5 mm MgCl 2 15 mm NH 4 SO 4 S2 Bovine Serum Albumin (BSA) 1.0 mg/ml in DEPC water 75 µl S3 β-nad 10 mm in DEPC water 30 µl Continued on next page v

6 Kit Contents and Storage, Continued cdna Synthesis Module, continued Vial Reagent Composition Quantity S4 1X RNase H Dilution Buffer 50 mm Tris-HCl, ph mm KCl 60 µl 10 mm MgCl mm EDTA 50 mm dithiothreitol 50 µg/ml BSA, ph % glycerol S5 Mussel Glycogen 2.0 mg/ml in DEPC water 60 µl S6 EDTA, ph M in DEPC water 12 µl S7 Phenol-Chloroform Phenol:chloroform:isoamyl alcohol (25:24:1) 0.1% 8-Hydroxyquinoline 2 x 1.0 ml S8 Ammonium Acetate 4 M in DEPC water 1.5 ml Autoclaved, RNase-free reaction vials Enzyme Module Reagents included in the Enzyme Module are described in the table below. Store at -20 C. Vial Reagent Composition Quantity E1 Test RNA (HeLa mrna) 0.3 µg/µl in DEPC water 18 µl E2 RNase Inhibitor 10 units/µl in DEPC water 7 µl E3 AMV Reverse Transcriptase 25 units/µl 12 µl E4 1 M Dithiothreitol (DTT) 1 M DTT in DEPC water 20 µl E5 E. coli RNase H 3 units/µl 7 µl E6 E. coli DNA Ligase 2.5 units/µl 7 µl E7 E. coli DNA Polymerase I 10 units/µl 15 µl E8 T4 DNA Polymerase 3 units/µl 7 µl vi

7 Accessory Products Additional Products Additional Reagents that may be used with this kit are available separately from Invitrogen. Ordering information is provided below. Product Amount Catalog no. FastTrack 2.0 mrna Isolation Kit 6 reactions K Micro-FastTrack 2.0 mrna Isolation Kit 20 reactions K Micro-to-Midi Total RNA Purification System 50 reactions Cytoplasmic RNA Reagent 100 ml TRIzol Reagent 100 ml EcoR I (Not I) Adapters 30 µg Random Primer 9 A 260 units Oligo dt/(not I) Unidirectional 15 µg N T4 DNA Ligase (1 unit/µl) 100 units AMV Reverse Transcriptase, Cloned 750 units RNase AWAY Reagent 250 ml vii

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9 Introduction Overview Introduction The Copy Kit is a cdna synthesis system for converting single-stranded RNA into double-stranded, blunt-end cdna. Blunt-end DNA adaptors may be ligated to cdna produced by the Copy Kit to improve efficiency of insertion into a plasmid or lambda phage vector. A test RNA sample is included to verify the activity of the components of the kit or become familiar with the Copy Kit procedure. The Copy Kit system uses the following process to generate blunt-end cdna from RNA (see Diagram on the next page): Conversion of single-stranded RNA to an RNA-cDNA hybrid by reverse transcription Conversion of the RNA-cDNA hybrid into double-stranded cdna using a method described by Gubler and Hoffman (see below) Conversion of the cdna to blunt-end form by treatment with T4 DNA polymerase The Gubler- Hoffman Method of cdna Synthesis The Copy Kit generates blunt-end cdna by methods described by Okayama and Berg (Okayama and Berg, 1982) and later by Gubler and Hoffman (Gubler and Hoffman, 1983). The following steps are involved to generate blunt-end cdna. Refer to the figure on the next page for an illustrated depiction of the cdna synthesis. 1. The AMV Reverse Transcriptase utilizes either oligo dt (Not I) or random primer to convert single-stranded RNA into an RNA-cDNA hybrid by reverse transcription. For more details on the primers, see next page. 2. The E. coli RNase H nicks the RNA while in RNA-DNA hybrid form. 3. The enzyme E. coli DNA polymerase I utilizes the fragments of nicked RNA as primers to synthesize the second strand of cdna. Any nicks in the double-stranded cdna are repaired by E. coli DNA ligase. This results in the formation of double-stranded cdna that retains a 3' overhang at the end of the first strand of cdna and/or a few nucleotides of RNA sequence at the 5' end of the converted strand. 4. The T4 DNA Polymerase converts the double stranded cdna blunt-end cdna. This is a prerequisite for adaptor addition. Continued on next page 1

10 Overview, Continued Diagram A schematic representation of the Gubler-Hoffman procedure for cdna synthesis is shown below. Modified Gubler-Hoffman Procedure mrna 5' mrna 3' cdna AAAAAAAAA(A) N AMV Reverse Transcriptase AAAAAAAAA(A) N TTTTTTTTTTTT Not I E. coli RNase H 5' mrna 3' cdna AAAAAAAAA(A) N TTTTTTTTTTTT Not I RNA E. coli DNA Polymerase I and E. coli DNA Ligase 5' cdna 3' cdna AAAAAAAAA(A) N TTTTTTTTTTTT Not I T4 DNA Polymerase 5' cdna 3' cdna AAAAAAAAA(A) N TTTTTTTTTTTT Not I Primers Oligo dt primers are most often used to generate full-length cdna clones. The oligo dt (Not I) primer is designed with an internal Not I site. Following adaptor addition, the cdna can be digested with Not I, purified, and cloned into an appropriate vector in the correct orientation for expression. Because it is a rare cutter, methylation of the cdna is not required. Internal priming with usersupplied oligonucleotides may also be used. Random primers are commonly used to generate smaller fragments of the gene, particularly when the 5' sequence of a large gene cannot be obtained by oligo dt priming. 2

11 Experimental Overview Experimental Outline If you are a first time user of the Copy Kit, read the entire manual before using the kit. It is important to perform each reaction exactly as described. A procedure checklist provided on page 11 can be used during the procedure by filling in one of the columns once a step is completed. The table below outlines the experimental steps necessary to generate doublestranded blunt-end cdna from your RNA sample. Step Action Page 1 Isolate RNA using a method of choice. 4 2 Perform first strand cdna synthesis 6 3 Perform second strand cdna synthesis 7 4 Generate blunt-end cdna 7 5 Ethanol precipitate the double-stranded, blunt-end cdna 8 Materials Supplied by the User Be sure to have the following items on hand before starting: Microcentrifuge kept at +4 C Sterile disposable Pasteur pipettes Water baths or heating blocks at 15 C, 42 C, 65 C, and 70 C Ice bucket, wet ice, and dry ice 80% ethanol in water made from high-purity 200-proof ethanol at -20 C α- 32 P-labeled dctp (if you wish to determine the cdna yield, see page 9) 3

12 Methods Isolating RNA Introduction You will need to isolate high-quality RNA using a method of choice prior to using this kit. Follow the guidelines provided below to avoid RNase contamination. General Handling of RNA When working with RNA: Use disposable, individually wrapped, sterile plasticware. Use only sterile, new pipette tips and microcentrifuge tubes. Wear latex gloves while handling reagents and RNA samples to prevent RNase contamination from the surface of the skin. Always use proper microbiological aseptic technique when working with RNA. You may use RNase AWAY Reagent, a non-toxic solution available from Invitrogen (see page vii) to remove RNase contamination from surfaces. For further information on controlling RNase contamination, see Current Protocols in Molecular Biology (Ausubel et al., 1994) or Molecular Cloning: A laboratory Manual (Sambrook et al., 1989). RNA Isolation The starting material can either be total RNA or mrna. You may isolate mrna or total RNA using the method of choice prior to using this kit. We recommend isolating mrna using the Micro-FastTrack 2.0 or FastTrack 2.0 mrna Isolation Kits. To isolate total RNA, we recommend TRIZOL Reagent or the S.N.A.P. Total RNA Isolation Kit. Ordering information for these kits is provided on page vii. Once you have isolated the RNA, check the quality of your RNA preparation. Important It is very important to use the highest quality of RNA possible to ensure success. Check the integrity and purity of your RNA before starting (see below). Checking the RNA Quality To check RNA integrity, analyze 500 ng of your RNA by agarose/ethidium bromide gel electrophoresis. You may use a regular 1% agarose gel or a denaturing agarose gel (Ausubel et al., 1994). For total RNA using a regular agarose gel you should see the 28S and 18S rrna bands. The 28S band should be twice the intensity of the 18S band. The 28S band should run at 4.5 kb and the 18S band should run at 1.9 kb. If you do not load enough RNA, the 28S band may appear to be diffuse. If you are using a denaturing gel, the rrna bands should be very clear and sharp. mrna will appear as a smear from 0.5 to 12 kb. If you do not detect any RNA, repeat RNA isolation. Be sure to follow the recommendations listed above to prevent RNase contamination. 4

13 cdna Synthesis Introduction Synthesize cdna with your RNA sample as the template using AMV Reverse Transcriptase. cdna synthesis is a multi-step procedure requiring many specially prepared reagents, which is crucial to the success of the process. Quality reagents (except ethanol and isopropanol) necessary for converting your RNA sample into double-stranded, blunt-end cdna are provided in this kit. To obtain the best results do not substitute any of your own reagents, including sterile water, for the reagents supplied with the kit. It is not necessary to monitor the reactions with tracer radionucleotides as the procedures and reagents are standardized in this kit. If you wish to determine the quality of your starting RNA and the yield and quality of the cdna synthesized, use labeled deoxynucleotide triphosphate in the reverse transcription reaction (see next page). A protocol is provided on page 9 to estimate the cdna yield. Use the procedure checklist provided on page 11 to prevent any errors from occurring during reagent additions. Before Starting 1. Equilibrate one water bath to 42 C and another to 65 C. 2. On ice, thaw red-capped vials RT1-RT6 from the cdna Synthesis Module. If there is a precipitate in the 5X RT Buffer (vial RT2), warm the solution to 42 C prior to use. If the precipitate persists, spin the tube briefly to pellet the precipitate. This will not affect the reaction. 3. Transfer the Enzyme Module to -20 C just prior to use. Remove the Enzyme Module from -20 C only when ready to use. Immediately after use, return the Enzyme Module to -20 C. 4. Remove Test RNA (HeLa mrna, vial E1) from the Enzyme Module and thaw on ice, if you wish to perform the control reaction. Continued on next page 5

14 cdna Synthesis, Continued First Strand cdna Synthesis 1. In RNase-free reaction vials, add the following on ice: Reagent Sample Tube Control Tube RNA Sample 2-5 µg -- Test RNA (HeLa mrna, vial E1)) µl Oligo (dt)/(not I) Primer (vial RT5) or Random Primer(vial RT6) 5 µl 5 µl Sterile Water (vial RT1) to 32.5 µl 12.5 µl 2. Mix the contents by tapping the tube and then centrifuge briefly to collect the contents of the tube. 3. Place the vials at 65 C for 10 minutes to denature the RNA secondary structure and then place the vials at room temperature for 2 minutes. 4. Centrifuge the tube briefly in a microcentrifuge at maximum speed. 5. Add the following to the tubes from Step 4: RNase Inhibitor (vial E2) 1.0 µl 5X RT Buffer (vial RT2) 10.0 µl 100 mm dntp (vial RT3) 2.0 µl 80 mm Sodium Pyrophosphate (vial RT4) 2.5 µl AMV Reverse Transcriptase (vial E3) 2.0 µl Note: 10 µci of 32 P-dCTP (3000Ci/mmol) can be added to the reaction if you wish to monitor first-strand incorporation (see page 9). 6. Mix the contents by tapping the tube and then spin briefly in a microcentrifuge at maximum speed. 7. Incubate the tubes at 42 C for 60 minutes. 8. Equilibrate a water bath to 15 C while the RT reaction is in progress. 9. Ten minutes before the end of the RT incubation, place vials S1-S4 and vial E4 on ice 10. Chill the first strand synthesis reaction on ice for 2 minutes and proceed to Second Strand cdna Synthesis, next page. Continued on next page 6

15 cdna Synthesis, Continued Second Strand cdna Synthesis 1. For each synthesis reaction, dilute the E. coli RNase H as follows: E. coli RNase H (vial E5) 1 µl 1 M DTT (vial E4) 1 µl 1X RNase H Dilution Buffer (vial S4) 8 µl 2. Mix the contents and place the tube on ice. 3. Add the following second strand reagents in order to the tubes containing 50 µl of the first strand reaction (Step 10, previous page). Second-Strand Buffer (vial S1) µl 1.0 mg/ml BSA (vial S2) 12.5 µl 10 mm β-nad (vial S3) 5.0 µl 1 M DTT (vial E4) 2.5 µl Diluted RNase H (0.3U/µl) 10.0 µl E. coli DNA Ligase (vial E6) 1.0 µl E. coli DNA Polymerase I (vial E7) 2.5 µl 4. Mix the contents by tapping the tube and briefly centrifuge in a microcentrifuge at maximum speed. 5. Incubate the reaction mixture at 15 C for 90 minutes. 6. Remove the tube from 15 C and incubate an additional 30 minutes at room temperature to complete the second strand synthesis. 7. Equilibrate another water bath to 70 C and place vials S5-S8 on ice. 8. Place the second strand reaction tubes at 70 C for 10 minutes. Centrifuge the tubes at maximum speed in a microcentrifuge. 9. Place the tubes at room temperature for 2 minutes and then place on ice for 2 minutes. Proceed to Generating Blunt-End cdna, below. Generating Blunt- End cdna 1. Add 1 µl of T4 DNA Polymerase (vial E8) to the tubes from Step 9, above. 2. Mix the contents by tapping the tube and centrifuge briefly in a microcentrifuge at maximum speed. 3. Incubate at room temperature for 10 minutes. 4. Add 2 µl of 0.5 M EDTA (vial S6) to stop the reaction. Proceed to Ethanol Precipitation, next page. Continued on next page 7

16 cdna Synthesis, Continued Ethanol Precipitation 1. Add 250 µl of Phenol-Chloroform (vial S7) to the tubes. Mix the contents by inverting the tube several times and centrifuge in a microcentrifuge at maximum speed for 5 minutes. 2. Carefully pipette the upper aqueous phase (contains double-stranded, blunt-end cdna) to a fresh RNase-free reaction vial. 3. Add 5 µl of Mussel Glycogen (vial S5). 4. Add 250 µl of 4 M Ammonium Acetate (vial S8) and 1 ml of 200-proof ethanol (stored at -20 C). Invert several times to mix the contents and place the tube on dry ice for 15 minutes. Note: This is a possible stopping point. Store the tubes at -20 C. 5. Centrifuge the reaction vial for 15 minutes in a microcentrifuge at maximum speed at +4 C. 6. Remove and discard the ethanol supernatant with a pipette. To avoid crosscontamination, use a new pipette for each cdna sample. Do not disturb the DNA pellet while removing the ethanol. 7. Add 500 µl of 80% ethanol and gently rock the tube twice. Centrifuge the tube at +4 C for 5 minutes in a microcentrifuge at maximum speed and carefully remove the ethanol. Centrifuge once more for 10 seconds to remove any traces of ethanol. 8. Store the double-stranded, blunt-end cdna at -20 C or -80 C. The Next Step The cdna generated with the Copy Kit is blunt-ended and ready for linker or adapter addition. For unidirectional cloning, digest the cdna with Not I and then add the linker or adapter. This will create a Not I overhang on the 3 end of the cdna, and allow cloning in the proper orientation for expression. Purification of cdna after linker addition is necessary to remove any unligated linkers or adapters prior to cloning. See page vii for ordering information on adapters. 8

17 Appendix Determining the Yield of First-Strand Synthesis Introduction Instructions are provided below to calculate the yield of your first-strand synthesis. Use extreme caution when working with radioactive material. Follow all rules regarding radiation safety. Contact your Safety Department for more information. Materials Supplied by the User Be sure to have the following items on hand before starting: 0.5 M EDTA Glass fiber filters Heat lamp (optional) 5% trichloroacetic acid (TCA) containing 20 mm sodium pyrophosphate 70% ethanol Forceps Scintillation counter Procedure 1. Label two Whatman GF/C glass fiber filters (one as "T" for "total" and the other as "I" for "incorporated") for each cdna sample to be analyzed. The "T" filters is used to measure the total amount of radioactivity in the reaction. The "I" filter is used to measure only the acid-precipitable radioactivity. 2. Transfer 2 µl of the first-strand cdna synthesis reaction from Step 10, page 6 to a new tube and add 1 µl 0.5 M EDTA and 47 µl water. Store the tube on ice. 3. Spot 5 µl of the diluted first-strand reaction from Step 2 above, onto each of the two labeled filters. Allow the filters to air dry at room temperature. 4. Prepare four beakers each containing ml of ice cold 5% TCA containing 20 mm sodium pyrophosphate. 5. Using forceps, transfer the "I" filter to one of the beakers. Swirl the filter in the acid solution for 2 minutes and then transfer it to the second beaker. Continue this successive washing with the two remaining beakers containing TCA/sodium pyrophosphate solution. 6. Transfer the washed "I" filter to a beaker containing 70% ethanol and keep the filter for 2-3 minutes. Air-dry the filter completely at room temperature or under a heat lamp. 7. Insert both the washed "I" and unwashed "T" filters into separate scintillation vials. Count both filters using a standard scintillation counter. Continued on next page 9

18 Determining the Yield of First-Strand Synthesis, Continued Calculations Calculate the amount of radiolabeled deoxynucleotide incorporated as follows: Proportion incorporated = cpm in washed filter cpm in unwashed filter Total cdna synthesized = total weight of nucleotides in the first-strand reaction X proportion incorporated For example: cdna was made using 5 µg of mrna. 2 µl of 100 mm dntps (25 mm of each nucleotide) or 200 nmole dntps was added to the reaction. Assume that the average molecular weight of deoxynucleotides is 330 ng/nmole then the total weight of nucleotides added is 6.6 x 10 4 ng. The filters were counted with the following results: Unwashed filters (total cpm) = 100,000 cpm Washed filters (incorporated cpm) = 5,000 cpm Proportion incorporated = 5,000 cpm = ,000 cpm Total synthesized = (0.05) X (6.6 x 10 4 ng) = 3.3 x 10 3 ng = 3.3 µg 66% of the starting mrna was converted to cdna. 10

19 Procedure Checklist for cdna Synthesis Introduction An abbreviated procedure checklist is provided below. Once a step is completed mark on the checklist. Each column corresponds to one cdna synthesis reaction that can be performed with this kit. Using the checklist will prevent any errors from occurring during reagent additions Step 2-5 µg of your RNA sample in water (15 µl of Test HeLa mrna, vial E1)!!!!! 5 µl Oligo dt (Not I) Primer (vial RT5) or Random Primer (vial RT6) Sterile Water (vial RT1) to 32.5 µl (for Test HeLa mrna add 12.5 µl)!!!!! Heat 65 C for 10 minutes!!!!! Incubate at room temperature for 2 minutes, briefly centrifuge Add the following first strand reagents to the tube:!!!!! 1 µl RNase Inhibitor (vial E2)!!!!! 10 µl 5X RT Buffer (vial RT2)!!!!! 2 µl 100 mm dntp (vial RT3)!!!!! 2.5 µl 80 mm Sodium Pyrophosphate (vial RT4)!!!!! 2 µl AMV Reverse Transcriptase (vial E3) Total reaction volume from first strand synthesis = 50 µl!!!!! Mix the contents and centrifuge briefly!!!!! Incubate at 42 C for 60 minutes!!!!! Chill on ice for 2 minutes For each synthesis, dilute the RNase H to 0.3U/µl as follows in a fresh tube :!!!!! 1 µl E. coli RNase H (vial E5)!!!!! 1 µl 1 M DTT (vial E4)!!!!! 8 µl 1X RNase H Dilution Buffer (vial S4)!!!!! Mix the contents and place the tube on ice To the first-strand reaction tube, add the following second strand reagents:!!!!! µl 1X Second Strand Buffer (vial S1)!!!!! 12.5 µl BSA 1 mg/ml (vial S2)!!!!! 5 µl 10 mm β-nad (vial S3)!!!!! 2.5 µl 1 M DTT (vial E4)!!!!! 10 µl diluted RNase H (diluted as above)!!!!! 1.0 µl E. coli DNA Ligase (vial E6) Continued on next page 11

20 Procedure Checklist for cdna Synthesis, Continued Step!!!!! 2.5 µl E. coli DNA Polymerase I (vial E7) Total reaction volume for second strand synthesis = 250 µl!!!!! Mix the contents and centrifuge briefly!!!!! Incubate at 15 C for 90 minutes!!!!! Incubate at room temperature for 30 minutes!!!!! Incubate at 70 C for 10 minutes!!!!! Incubate at room temperature for 2 minutes!!!!! Incubate on ice for 2 minutes!!!!! Add 1 µl T4 DNA Polymerase (vial E8), mix and centrifuge briefly!!!!! Incubate at room temperature for 10 minutes!!!!! Add 2 µl 0.5 M EDTA (vial S6), 250 µl Phenol-Chloroform (vial S7), mix the contents and transfer the top aqueous layer to a new tube!!!!! Add 5 µl Mussel Glycogen (vial S5)!!!!! Add 250 µl 4 M Ammonium Acetate (vial S9), 1 ml 100% ethanol, freeze on dry ice for 15 minutes!!!!! Centrifuge for 15 minutes at +4 C and remove the ethanol!!!!! Add 500 µl 80% ethanol, mix the contents, centrifuge for 5 minutes at +4 C, and carefully remove the 80% ethanol!!!!! Centrifuge briefly and remove any traces of ethanol!!!!! Store the double-stranded, blunt-end cdna at -20 C or -80 C 12

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22 Technical Service, Continued Emergency Information In the event of an emergency, customers of Invitrogen can call the 3E Company, 24 hours a day, 7 days a week for disposal or spill information. The 3E Company can also connect the customer with poison control or with the University of California at San Diego Medical Center doctors. 3E Company Voice: Limited Warranty Invitrogen is committed to providing our customers with high-quality goods and services. Our goal is to ensure that every customer is 100% satisfied with our products and our service. If you should have any questions or concerns about an Invitrogen product or service, please contact our Technical Service Representatives. Invitrogen warrants that all of its products will perform according to the specifications stated on the certificate of analysis. The company will replace, free of charge, any product that does not meet those specifications. This warranty limits Invitrogen Corporation s liability only to the cost of the product. No warranty is granted for products beyond their listed expiration date. No warranty is applicable unless all product components are stored in accordance with instructions. Invitrogen reserves the right to select the method(s) used to analyze a product unless Invitrogen agrees to a specified method in writing prior to acceptance of the order. Invitrogen makes every effort to ensure the accuracy of its publications, but realizes that the occasional typographical or other error is inevitable. Therefore Invitrogen makes no warranty of any kind regarding the contents of any publications or documentation. If you discover an error in any of our publications, please report it to our Technical Service Representatives. Invitrogen assumes no responsibility or liability for any special, incidental, indirect or consequential loss or damage whatsoever. The above limited warranty is sole and exclusive. No other warranty is made, whether expressed or implied, including any warranty of merchantability or fitness for a particular purpose. 14

23 Product Qualification Quality Control Each component of the Copy Kit is lot qualified for optimal performance. For each lot, two cdna synthesis reaction (one using the Oligo(dT) Not I primer and the other using the random primers) are performed on HeLa mrna. Second strand cdna is checked for the presence of known housekeeping genes (actin and clathrin) to ensure proper representation and full-length synthesis. The cdna is size selected (1000 bp) and both a unidirectional and a bidirectional library is constructed. Each library must adhere to the following specifications: 1 x 10 6 primary recombinant 90% of recombinant contain an insert Average insert size of 1.0 kb Vector background was ~ 1% 15

24 References Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., and Struhl, K. (1994). Current Protocols in Molecular Biology (New York: Greene Publishing Associates and Wiley- Interscience). Gubler, U., and Hoffman, B. J. (1983). A Simple and Very Efficient Method for Generating cdna Libraries. Gene 25, Okayama, H., and Berg, P. (1982). High-Efficiency Cloning of Full-Length cdna. Mol. Cell. Biol. 2, Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual, Second Edition (Plainview, New York: Cold Spring Harbor Laboratory Press) Invitrogen Corporation. All rights reserved. 16