The chimeric molecules generated during PCR are really bad for our study on recombination, since the chimeras look exactly like recombination. I wonder what and how (would be better if protocol is available) we can reduce producing PCR chimeras.
1) Maximize elongation time: Minimizes the formation of incomplete elongation products that function as primers in subsequent PCR cycles.
2) Use a highly processive DNA polymerase such as Platinum Pfx to also reduce the chance of incomplete elongation products.
3) Minimize the number of total PCR cycles: reduces the formation of chimeras.
4) Use low input DNA amounts in the PCR: has as large an effect in papers of increasing the elongation time.
Good luck!
in addition to Chris's answer use primers with a high annealing temperature. all enzymes make more mistakes when amplifying at the wrong temperature ( you often see more clone sequence errors near the primer ends of an amplimer) so primers annealing at close to the extension temperature of the enzyme are best. The enzyme will be amplifying even at the lower temperature of annealing the primer even if it does so at a slightly slower rate
To add to both of the above answers:
1. Try a 2 step PCR in which an initial denaturation cycle is followed by a combined annealing/Extension cycle: Thus, Make primers with Tms of about 68-70C and try the annealing extension step @ the Tm (i.e 70C) or Tm -2 i.e. 68C. In many instances this leads to more specific PCR amplifications and is commonly deployed as a trouble shooting aid relative to standard 3 step PCR for that reason. What is more, many of the new highly processive polymerases like phusion or platinum pfx operate perfectly efficiently @ the Tm, unlike WT taq
2. I would caution against using higher concentrations of primer, as like too many cycles (>40) this encourages the amplification of secondary non specific products
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