Categories See All
Search Articles
Recent Articles
Recent Comments
-
How to avoid contamination...
By muhammad ilyas, Apr 26 2013 04:15 AM -
How to avoid contamination...
By Anoop Bhaskaran, Feb 07 2013 01:44 AM -
How to convert g into rpm?
By Matthijs, Jan 26 2013 01:04 PM -
Handling RNA - How to avoid...
By Myworkismyplay, Dec 26 2012 09:31 PM -
How to get rid of PCR prim...
By Myworkismyplay, Dec 26 2012 09:25 PM -
How to avoid contamination...
By Myworkismyplay, Dec 26 2012 09:17 PM
How to get rid of PCR primer dimer
1) Try the DMSO up to 5%. Some polymerases are ok with DMSO and others aren't. You never know until you try.
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.
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.













1 Comments
Myworkismyplay
Dec 26 2012 09:25 PM