A standard 3-step cycling profile was used, 95°C for 1 min, followed by 35 cycles of 95°C for 15s, 60°C for 15s and 72°C for 10s. These were set-up at room temperature and subsequently 5μl of the final product was run on a 1% agarose gel and stained with ethidium bromide.
RESULTS AND DISCUSSION
At very high bacterial concentrations, PCR is inhibited, but at lower concentrations (under normal working concentrations) the cell debris is not very inhibitory to either MyTaq Mix or Supplier S mix (fig. 1), suggesting that cell debris is not normally a major contributing factor to poor colony PCR.
Both LB (fig. 2A) and agar (fig. 2B) are inhibitory at high concentrations, particularly with larger PCR fragments; however MyTaq was able to tolerate much higher concentrations than Supplier S. MyTaq Mix can be used to successfully screen recombinant clones starting with crude colony preparations or suspensions, in a highthroughput method. The mix is robust enough to amplify a wide range of fragment sizes using different primer pairs, for amplicons of up to about 3kb (fig. 3).
MyTaq is evidently a highly robust and versatile polymerase and together with a novel buffer, delivers high performance in chemically complex reaction conditions. The result is superior tolerance to a wide range of common PCR inhibitors, which results in unsurpassed performance in colony PCR. An added advantage is that MyTaq can also be used to directly screen overnight LB cultures, so minimizing the cost of plasmid preparation of uninteresting clones.
The use of MyTaq HS, a hot-start polymerase, also allows convenience of leaving the PCR reaction at room temperature before processing, which can be advantagous when working with a large number of colonies.