BC016 – Optimizing TRIM PCRs: Annealing temps, cycle number, and DMSO concentration

Submitted by Brittnee Crane

The cDNA treated with 5 and 50 ng/μL of LPS and that was mentioned in BC004a was used to make PCR mixtures for TRIM31, TRIM69, TRIM22, TRIM56, and TRIM6. Primers that were previously designed and ordered were used in the reaction. Two different reactions were made for each: one with the 5 ng/μL LPS cDNA and one with the 50 ng/μL LPS cDNA, both containing 5% DMSO. The Q5 Master Mix settings were used in the thermocycler, but the number of cycles was increased from 30 cycles to 37 cycles, and the extension time was increased from 1 minute to two minutes, and the annealing temperature was changed to 53°C. The decrease in the annealing temperature proved to be ineffective.


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