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Another thing you may want to consider is making your primers long enough. When you introduce restriction sites and tags, you are introducing a lot of sequence that has no complementarity. If the instability introduced by the non-complementary part of the primer is higher than the stability provided by the complementary part of the primer, you will not get any amplification no matter at what temperature you anneal.
Many times you can save yourself time by designing multiple primers and testing them together rather than performing a lot of troubleshooting on a single primer set. Primer design can be very tricky, and the rules about how much non-complementary primer you can have and still get PCR amplification are sketchy at best.
#28664 PCR Cloning-large primers
Posted
on 08 July 2009 - 07:28 AM
#35622 PCR-how to set 'Extension: ramping from 55 to 72 for 5min'
Posted
on 07 September 2009 - 10:46 PM
fortunate, on Sep 4 2009, 02:08 AM, said:
Swanny, Thank you very much! Hope to see you again here.
swanny, on Sep 1 2009, 11:34 PM, said:
From what you've said, I presume you are working with different primers in the same sample, the same primers in a few different samples (with varying degrees of homology), and/or you are trying to get different regions of the same template DNA amplified using common primers, or something else like that, right?
Thank you so much for your help! I was doing knockout genotyping using mouse tail DNA. Yes, I used two pairs of primers, one for wild type, one for knockout.
Not knowing which machine you are using, I can't start to give great detail about how to program this ramped extension.
I am using MJ Mini personal thermal cycler from Bio-Rad. What you could do is to have 4 or 5 extension steps in each cycle. Each step would last for 60 sec, say, then increase by a degree or two. The range for the extension step is pretty big, going from 55 (which will give almost no polymerase activity) to 72. Could you explain a bit more what is the purpose to do this kind of extension? What will be the difference if I just use a fixed temp like 72 'C?
Can you give us any more details of the project?
I was using a kit from GeneScript http://www.genscript...out_Enzyme.html. They mix the buffer with their polymerase. It is the kit's recommedation that I use that Extension temperature range and 5min. My first PCR trial took over 7 hours!
Thank you so much for your help! I was doing knockout genotyping using mouse tail DNA. Yes, I used two pairs of primers, one for wild type, one for knockout.
Not knowing which machine you are using, I can't start to give great detail about how to program this ramped extension.
I am using MJ Mini personal thermal cycler from Bio-Rad. What you could do is to have 4 or 5 extension steps in each cycle. Each step would last for 60 sec, say, then increase by a degree or two. The range for the extension step is pretty big, going from 55 (which will give almost no polymerase activity) to 72. Could you explain a bit more what is the purpose to do this kind of extension? What will be the difference if I just use a fixed temp like 72 'C?
Can you give us any more details of the project?
I was using a kit from GeneScript http://www.genscript...out_Enzyme.html. They mix the buffer with their polymerase. It is the kit's recommedation that I use that Extension temperature range and 5min. My first PCR trial took over 7 hours!
As for the 5 minute extension, my first guess would that the enzyme is not very fast at the lower temperatures (anything below 65, as memory serves), so for the first couple of minutes your DNA polymerase is crawling along the template. Then as the temperature rises above 65, the reaction rate increases. If your primers have similar Tms, you might be able to shorten the extension time, and also reduce the temperature range.
What extension times and temp do you use for single primer sets? You should use the longer extension time, and lower temperature as your first parameters.
