PCR - BioWiki Knowledge Base http://www.protocol-online.org/forums/page/index.html/_/pcr/ Thu, 09 Feb 2012 13:53:03 +0000 43200 How to avoid contamination in PCR? http://www.protocol-online.org/forums/page/index.html/_/pcr/how-to-avoid-contamination-in-pcr-r16
I have followed the following with good results...
  • Prepare your master mix in a separate room from your current location, somewhere PCR is not the main practice... Be sure to have a separate lab coat, gloves, tubes, pipette tips tobe used only in that clean room.
  • Use a separate aliquot of DEPC water stock for each round of PCR
  • Prepare your mix in a hood with laminar flow. Decontaminate it with bleach, alcohol, RNAse, DNase, etc... Be sure to UV-irradiate pipettes, pipette tips, tubes, racks, gloves, and also your aliquots of water and PCR buffer... before the procedure.
  • Use a different pipette tip when pipetting all your reagents, even the same master mix to each tube
  • Keep your tubes closed during the procedure, even your master mix tube. Be sure that your tubes are closed when discarding the pipette tip!!! Aerosols are dangerous!!! Open the tubes only when necessary.
  • More important... schedule your PCR when not handling plasmids!!!

Hope this help...
Good luck]]> Sat, 07 Aug 2010 20:51:01 +0000 c45147dee729311ef5b5c3003946c48f How to get rid of PCR primer dimer http://www.protocol-online.org/forums/page/index.html/_/pcr/rt-pcr/how-to-get-rid-of-pcr-primer-dimer-r15
2) Try a two step PCR. There is a paper in Biotechniques about a two step PCR reaction but I don't have the reference with me since I'm at home and it's in lab. Its worth looking up. Basically you've got template plus forward primer in one tube and template plus reverse primer in another tube and then do between 1-15 rounds of PCR. Then combine the contents of the two tubes and do your normal 25 rounds of PCR. Sometimes this allows the primer to bind to the template effectively and allow you to get some initial transcripts which will then bind to each other when you combine the contents of the two tubes.

3) If you're not using a polymerase for high GC content, then get one. It will save you a lot of hassle. It won't solve your problem but it will help you narrow down things when troubleshooting. KOD XL polymerase (novagen) isn't sensitive to DMSO up to 5% and it helped me get all of my mutants but not without a lot of tries just because the GC content of primer and template were over 60%. Some people like the quik change kit by stratagene but I never got it to work, which is really expensive when its $600 a kit for 30 rxn or something like that. To each his own.

4) I wouldn't go below 55 deg for an annealing temp. 50 might be fine but below that you're pushing your luck with trying to have a high enough temp to resolve any hairpin structures which are common in high GC rich primers not to mention possibly decreasing the specificity of matching primer and template.

good luck! it takes a lot of troubleshooting and its hard to get consistent results.]]> Sat, 07 Aug 2010 20:48:39 +0000 2b44928ae11fb9384c4cf38708677c48