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Michelle Chew, BDGP, Lawrence Berkeley National Labs
Reagents for one 14uL Rxn:
10X Advantage Buffer 1.4uL 10 mM dNTPs 0.3uL 20uM PM001a 0.225uL 20uM PM002a 0.225uL Template 3.0uL from 3:150 dilution in H2O from Original bacterial cDNA frozen stock plate Advantage Taq (5U/uL)* 0.15uL ddH2O 8.7uL
95C 1 min 95C 0.5min 73C-->63C* 0.75min Touchdown for 5 cycles 68C 3 min +0.2min [the annealing temp. was decreased by 2C for 5 cycles] 95C 1 min 63C 0.75min Auto extend for 28 cycles** 68C 4 min + 0.1min 68C 15 min 4C soak PM001a Primer 5'- GTCGACGTTAGAACGCGGCTAC - 3' PM002a Primer 5'- GGGTTAAATTCCCGGGTACTGC - 3'
Advantage cDNA Polymerase Mix (50x) (#8417-1) Available from CLONTECH Laboratories, Inc.
We have also successfully used DyNAzyme EXT DNA Polymerase (1U/uL) (F-505S) with EXT Buffer (F-514) made by Finnzymes and available from MJ Research, Inc. (1-888-662-2566)
We used the same amount of DyNAzyme per reaction (0.15uL) despite the lower unit concentration to size our cDNAs and it works fine. Although for longer PCR products, you might consider raising the volume of DyNAzyme used per reaction.
*The annealing temperatures were determined using nearest neighbor method. We did notice that sometimes we saw spurious bands so the annealing temperature was raised to 66C to help get rid of unwanted short products.
**You may also want to increase the number of cycles to 35 if you still cannot PCR the longest cDNAs. rev. 8.23.00