TM of primers 58.4 and 56.4
I don't know anything about annealing temperature, please guide me to find appropriate annealing temperature.
Thanks.
5 degrees under TM is fine, would be something like 52 degrees here
Try at 56 and 58 deg Centigrade. Most of the primers work at this temp with 1.5mm Mgcl2 Concentration.
Other option is use a gradient in the annealing temperature (Ta) (51 to 60¤C used to work). If you have a degenerated primer, not only have one primer with a defined Ta, you have a population with different Ta.
Hi
Thanks all
What does would be if I count average of them and then use 8 degree of average? is this useful?!
it would work but the annealing temperature would be so low that the primers would anneal all over the genome and the product would be really messy. You should not just take an average temperature as the lower annealing primer might not stick at all and there would be no amplification. If you do not have a gradient pcr machine I would try 2c lower than the tm of the lowere annealing primer just to get a product and if it is dirty then increase the annealing temperature in increments of 2c. The primers are in very large excess so a lot of primer will anneal even above tm . Understand that calculation of tm is possible in many different ways and can vary greatly for the same primer when calculated by different companies and is only a very approximate guide to help experimentation
Hi dear Rutland
Thank you for your kindly guidance
Yes I think you are right. I'll do your protocol.
Hi dear friends
I used gradient program from 51 to 57 in six repeat to three of my isolates.
These are their results and pics. I think this is primer dimer problem.
Do you have idea to decrease their unspecific bands?
can you give us some details like primer sequence and pcr reaction mix and cycling conditions please Kamal. Ideally also the od260/280 ratio and amount used in the pcr of a typical sample
You can test your primers here:
http://www.ncbi.nlm.nih.gov/tools/primer-blast/
Just copy-paste both primers, select nr database. Then first don't select any taxa. Second round you may select your target taxa.
I think it's hard to see anything from your pictures. And what's target size (or product size = target plus primers)?
http://www.ncbi.nlm.nih.gov/tools/primer-blast/
Dear Eero
Thank you for your kindly guidance
They are degenerate primer and I tested them in NCBI
I detect 200bp size and 400 bp.
Those pictures are my real pics. I don't know what you mean. But in this site all the researchers are free to ask any questions and research problems...
By the way, thank you for your guidance, but primers have lower melting temperature. I used gradient PCR from 56 to 62c, however it did not work upper also.
Be happy and make happy
kamal
Hi Dr. Burzynski
At the first be happy and make happy mean best wishes.
I used gradient bt upper than 57c that wasn't work it is that pic is from 58c to 62c. last single band is positive control for electrophoresis.
Then I used 2 degree lower and upper Tm it is second pic.
I think I should try your advice. thank you dr. I'll report my rsult.
best wishes
kamal
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