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Position: Discussion forums > Bioscience biotechniques discussion forums > Molecular biology troubleshooting forum
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Annealing primers
Posted by: AcetheISS (IP Hidden, New member, 5)
Date: June 11, 2005 11:13AM
Hi,
I have a problem annealing 6 primers. They are about 35bp each, and total 108 bp long (Three of them are complementary to the other three). A portion of one primer is spanning a portin of its complementary primer, and the other portion is spanning a portion of another primer.. hope it's clear enough.. it's hard to explain. Anyways, the annealing does not seem to work properly because it is too small on a gel. I annealed them at 75C.. It is too high for annealing?? It's so frustrating when something doesn't work when it should. Any thoughts will be appreciated! Catherine
Re: Annealing primers
Posted by: Beveren (IP Hidden, Unregistered user, )
Date: June 11, 2005 10:20PM
Why don't synthesize the whole length oligo (sense and antisense) and aneal them?
Re: Annealing primers
Posted by: AcetheISS (IP Hidden, New member, 5)
Date: June 12, 2005 02:17PM
Good reasoning, however: it is very difficult to synthesize very long oligos accurately and in good yield (each base loses maybe 1 or 2% in the yield), so the maximum we can order is about 50 bases. Too bad!
Should I try a lower annealing temperature?? Confusing..
Re: Annealing primers
Posted by: 5'-ATCG (IP Hidden, Unregistered user, )
Date: June 13, 2005 05:05PM
Custom synthesize gene (not oligo) only cost ~$1.50 per base, so total will cost you between $150-$200 with 100% guaranteed sequency accuracy. Easy, fast, cheap and no annealing temp. headacde........
Re: Annealing primers
Posted by: femmeauburn (IP Hidden, Advanced member, 115)
Date: June 14, 2005 12:32PM
Olgios can be synthesized in excess of 150 bases with sufficient quantities obtained by PAGE purification for successful PCR. However the longer the olgio the greater the chance of accumulaed errors, which can be screened for using a melting curve. You may wish to custom synthesize the gene as 5' suggests. But if you have enough sample left: Calculate the Tm of your primers. Typically the annealing temperature chosen is 5C below the lower Tm of the primer pair. If you do not know the Tm, a general rule is to start with 54C as the annealing temperature.
Re: Annealing primers
Posted by: fred_33 (IP Hidden, New member, 2)
Date: June 21, 2005 11:51AM
hi
usually when i anneal primers i heat the whole prep to 95° and let it cool slowely on the bench. It works properly. But i would strongly recommend you as Beveren did to order the whole primier. It's not that expensive and will be more comfortable.
Re: Annealing primers
Posted by: kumarsysu (IP Hidden, New member, 9)
Date: August 14, 2005 09:06PM
should the anealing temp. be low as much as 2 to 4 degree centigrade from the lowest Tm of the two ( forward and reverse) primers.
Re: Annealing primers
Posted by: femmeauburn (IP Hidden, Advanced member, 115)
Date: August 15, 2005 03:33PM
Typically the annealing temperature chosen is 5C below the lower Tm of the primer pair. 2 to 4 degrees lower should also be just fine.
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