Transcription of PCR Standard Protocol (with Taq polymerase)
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PCR Standard Protocol (with Taq polymerase)
PCR Standard Protocol (with Taq polymerase) H. Judelson WARNING: Contamination can be a major problem, unless you are careful. Common routes for contamination include using the same pipette to set up a reaction and load products on a gel, or using the same water for PCR and other activities such as restriction digests. Reagents used for PCR should be pipetted using filtered tips to reduce contamination. Aliquotting reagents will help ensure that they are clean. STEP 1: Create primer sequences based on the template, taking note of the melting temperatures (Tms). Program the PCR machine (see step 4). Typically, PCR is done with annealing temperatures 5C below the Tm, but the optimal temperature must be determined empirically. Most annealing temperatures are between 50 and 60C, for oligos that are 18-22 nt. Be careful: some oligo programs show Tms for several salt concentrations, not all of which are used for PCR (which is typically done at 50-60 mM depending on the enzyme system).
COLONY PCR (for screening E. coli colonies) -Using the same recipes as above, fill tubes with 25 µl PCR mix. -Pick up a TINY part of the colony using a toothpick or pipette tip and place in the PCR tube.
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