Human Placenta PCR-Ready cdna Kit Cat No. PC-40013: 30 reactions

Transcription

1 780 Dubuque Avenue So. San Francisco, CA 94080, U.S.A. Tel: (800) / Fax:(650) mbi@maximbio.com Human Placenta PCR-Ready cdna Kit Cat No. PC-40013: 30 reactions INSTRUCTION MANUAL *These products are designed and sold for use in the Multiplex PCR (MPCR) covered by patent # 5,582,989. Use of the MPCR process requires a license. A limited, non-automated research field license under the patent to use only this amount of the product to practice the MPCR process is conveyed to the purchaser by the purchase of this product. The Polymerase Chain Reaction (PCR) process is covered by patents owned by Hoffman-LaRoche. Use of the PCR process requires a license. A license for diagnostic purposes may be obtained from Roche Molecular System. A license for research may be obtained by the purchase and the use of authorized reagents and DNA thermocyclers from the Perkin-Elmer Corporation or by negotiating a license with Perkin-Elmer. This product is intended for research use only and not for diagnostic purposes. ID-M10183 Revised August 5, 2002

2 I. INTRODUCTION The products listed in this manual are for research use only. They are not for diagnostic or therapeutic use in humans or animals. Experiments performed using these products should conform to the NIH Guidelines for Research Involving Recombinant DNA Molecules or other applicable regulations or guidelines. It is the responsibility of the researcher to abide by such guidelines. These products are designed and sold for use in the Polymerase Chain Reaction (PCR) process covered by patents owned by Hoffman-LaRoche. Use of the PCR process requires a license. A license for diagnostic purposes may be obtained from Roche Molecular System. A license for re- search may be obtained by the purchase and the use of authorized reagents and DNA thermocyclers from the Perkin- Elmer Corporation or by otherwise negotiating a license with Perkin-Elmer. Certain reagents indicated for use in this manual are hazardous. The researcher is cautioned to exercise care with these reagents and ith the equipment used in these procedures, strictly following manufacturers safety recommendations. Disposal of waste organics, acids, bases, and radioactive materials must comply with all local state, federal, or other applicable regulations. II. COMPONENTS PC x50µl PCR Rxns Kit Component Volume PCR-Ready Human Placenta DNA: 1ng/µl 30 µl 2X PCR Buffer 1250µl Control Human beta-actin primers 20µl (BAC-1008/1004, 1OD/ml) ddh 2 0 (DNase free) 1.0 ml Components Required But Not Supplied Taq DNA Polymerase Gene-Specific PCR Primers mrna Source Human Placenta, pooled from 7 Caucasians, ages cdna Storage Buffer 1mM Tris-HCl (ph 8.0) and 0.1mM EDTA NOTE: Avoid multiple freeze/thaw cycles Shipping Condition Blue ice 2

3 III. OVERVIEW The PCR-ready cdna System is a set of pre-made cdna reagents intended for researchers to conduct amplifiatin with gene-specific oligonucleotide primers by the PCR method 2,3. Two control amplification primers (beta-actin specific primers) are included in the system to verify the performance of the RT-PCR amplification. Please note that the PCR-ready cdna System does not include Taq DNA polymerase or the reagents required for cloning. First strand cdna sythesis is initiated at the poly (A) tail of mrna using the oligo-dt anchor primer. The first single strand cdna molecules are then converted into double strand cdna using Gubler and Hoffman methods 1. The double stranded cdna has been extracted with phonol/chloroform and precipitated with ethanol. cdna is resuspended in 10mM Tris-HCl (ph 8.0) and 1mM EDTA. Amplification of a Target cdna Amplification of a target gene using cdna provided in the PCR-ready cdna System requires priming with two oligonucleotides and either Taq or AmpliTaq DNA Polymerase (Perkin-Elmer Cetus). These primers may contain a restriction endonuclease site portion (adapter region) that enables RT-PCR products to be cloned rapidly & efficiently. Cloning of Amplification Products Conventional cloning methods that typically involve end-repair and blunt-end cloning can be problematic for amplified products 4,5,6. Although these methods can be performed with the RT-PCR products, T/A Cloning Sysem from Invitrogen provides a rapid and efficient method for cloning 5 & 3 RACE products 7,12. COMPARISON OF MPCR WITH RPA Another approach to cloning is to digest the RT-PCR product using one of the restriction endonuclease sites designed into one of the PCR primers. Design of the Gene-Specific Primer The overall degeee of success attained with the RT-PCR will be determined in part by the gene specific primers (GSP). In general, all primers used would both have a Tm around 68 C. It should be noted that in cases where only limited peptide sequence information is available, a degenerate GSP may be prepared and used in this PCR-ready cdna system. Positive Control Primers The primer pair BAC-1008/1004 is included in this Kit, which can be used to amplify a 303 bp of human beta-actin gene. BAC-1008: 5 -GCC AAC CGC GAG AAG ATG ACC-3 Tm = 72 C Ta = 64 C (predicted annealing temperature) BAC-1004: 5 -CTC CTT AAT GTC ACG CAC GAT TTC-3 (In Exon 3) Tm = 67 C Ta = 60 C (predicted annealing temperature) 1 GCCAACCGCG AGAAGATGAC CCAGATCATG TTTGAGACCT TCAACACCCC AGCCATGTAC 61 GTTGCTATCC AGGCTGTGCT ATCCCTGTAC GCCTCTGGCC GTACCACTGG CATCGTGATG 121 GACTCCGGTG ACGGGGTCAC CCACACTGTG CCCATCTACG AGGGGTATGC CCTCCCCCAT 181 GCCATCCTGC GTCTGGACCT GGCTGGCCGG GACCTGACTG ACTACCTCAT GAAGATCCTC 241 ACCGAGCGCG GCTACAGCTT CACCACCACG GCCGAGCGGG AAATCGTGCG TGACATTAAG 301 GAG FIGURE 1. The Positive Control Amplicon Sequence. 3

4 IV. METHODS Amplification of Target cdna Protocol. The cdna from the PCR-Ready cdna Kit can be amplified directly using the PCR method. 1. Add the following components to a fresh 0.5-ml microcentrifuge tube: GSP PCR β-actin PCR Component Volume (µl) Volume (µl) 2X PCR Buffer Sterilized, distilled H 2 O PCR-Ready cdna* 1 1 Gene Specific Primers (GSP): 2µM each 5.0 or beta-actin primers: 2µM each 5.0 Taq DNA polymerase (2-5 U/µl) * More or less cdna can be used here depending on the copy number of your specific gene. 2. Mix gently. Collect the reaction briefly by centrifugation. Hot Start PCR is highly recommended here 8,9. 3. Depending on machine, mineral oil may be required here. See machine manual for details. 4. Perform rounds of PCR amplification, using the protocol accompanying AmpliTaq (Perkin-Elmer Cetus) or Taq DNA polymerase. The cycling protocol should be optimized for each sequence to be analyzed. An example of the profiles optimized for the beta-actin gene is given as follows: Temperature Time Cycles 94 C 3 min 1 cycle 94 C 1 min C 1.5 min cycles 72 C 10 min Soak at C 5. Analyze 10 ml of the amplified sample, using agarose gel electrophoresis and ethidium bromide staining, and the appropriate molecular size standards. If the positive control primer (BAC-1008/1004) is used, a 303 bp band will be visible. 4

5 TROUBLESHOOTING Observation Possible Cause Recommended Action No bands after electrophoretic analysis of amplified products. PCR Parameter error or PCR primer problem. 1. Use the control primer to verify PCR performance and parameters. 2. Re-check GSP primer design and QC data 3. Use more cdna or increase the cycle number Target mrna contains strong transcriptional pauses. Increase the Tm of the GSP primers; increase the PCR annealing temperature. Unexpected bands after electrophoretic analysis of RT-PCR products. Contamination by genomic DNA Spurious priming in the PCR Pre-treat RNA with DNase Vary the parameters of the PCR reaction as described by Perkin- Elmer Cetus. Incorporate a hot start step 8,9 Poor Cloning efficiency Inefficient ligation Increase the incubation time in the ligation reaction; Ensure the removal of dntps prior to ligation Poor restriction endonuclease digestion due to residual bound Taq DNA polymerase. Treat the PCR with proteinase K 10,11 5

6 PRECAUTIONS AND STORAGE Storage 1. Store all Kit components at -20 C. Under these conditions components of the kit are stable for 1 year. 2. Isolate the kits from any sources of contaminating DNA, especially amplified PCR product. 3. Do not mix kit components that are from different lots. Each lot is optimized individually. REFERENCES 1. Gubler, U. and Hoffman, B. (1983) Gene 25, Saiki, R.K., Scharf, S., Faloona, F., Mulis, K.B., Horn, G.T., Erlich, H. A., and Arnheim, N. (1985) Science 230, Saiki, R.K. Gelfand, D.H., Stoffel, S., Scharf, S. J., Higuchi, R., Horn, G.T., Mullis, K.B., and Erlich, H.A. (1988) Science 239, Scharf, S.J. (1990) PCR Protocols: A Guide to Methods and Applications (Innis, M.A., Gelfand, D.H., Sninsky, J.J., and White T.J., eds.) p84, Academic Press, San Diego. 5. Bhat, G.J., Lodes, M.J., Myler, J., and Stuart, K.D. (1991) Nucleic Acids Res. 19, Shuldiner, A.R., Scott, L.A. and Roth, J. (1990) Nucleic Acids Res, 18, Clark, JM. (1988) Nucleic Acids Res. 16: Mullis, K.B. (1991) PCR Meth, and Appl. 1,1. 9.Chou, Q., Russel, M., Birch, D.E., Raymond, J. & Bloch, W. (1992) Nucleic Acids Res. 20, Sambrook J., Fritsch, E.F., and Manaitis, R. (1989) Molecular Cloning: A laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York. 11.Browe, J. S., Cooper, H.J., Smith M.A., Sims, J.J., Parker, D., and Gewert, D. (1991) Nucleic Acids Res. 19, Mead, D. et al. (1991) Bio/Technology 9: