Sigma-Aldrich
REDTaq ReadyMix PCR Mix
R2523
Our project aimed at isolating homologs from genomic and cDNA of a plant whose DNA template was particularly difficult to work with. With a specific and difficult target to amplify the homologs, especially whose genome sequence data is not available, it was a task. Also to add to it, we were setting up and standardizing PCR for the first time in our lab. With all these challenges, it was particularly easy to work our way out with REDTaq PCR Mix. It does not require a lot of standardization and the results are reproducible every time. It is economical especially for start- up labs and Under-graduate and Post- graduate programs. Moreover, its red color also saves us the time in ensuring all the reagents are in the tube and not on the lid. It works best with both genomic DNA and cDNA as well.
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Polymerase Chain Reaction (PCR) for amplification of DNA
Purified Genomic DNA and cDNA from Plant
REDTaq PCR Mix basically has all the components required in a PCR, except the primers, template and water. Therefore, the following protocol was used by us for standardization of PCR: REDTaq PCR Mix 12.5microlt (Half reaction) F.Primer(10pmoles/microlt) 1microlt R.Primer(10pmoles/microlt) 1microlt Template(DNA-0.5microg/microlt; cDNA-50ng/microlt) 2microlt Water 8.5microlt) PCR Cylces: Denaturation 94degree 1min Annealing 50-60degree 2min Extenstion 72degree 3min Final extention 72degree 10min No of cycles- 35
Multiplex PCR works very well; Try to use half reactions- they work better than full reactions
The PCR amplification results were reproducible. With little standardization in annealing temperature, single band was obtained. We have worked with genes as long as 1Kb to 0.3Kb and it has given us very good quality bands, with minimum background smear and little primer-dimers. The sequencing of the PCR products in most cases have given a good quality chromatogram, minimum noise and more number of good quality base information.
None
Easy to standardize and reproducible results
Long duration of PCR
Best PCR mix for standardization, economical, and good quality results