primer Tm is too high, how tu get pcr product - help! (Oct/14/2010 )
I am cloning a gene fragment by pcr.But I cannot get pcr product.the primers:Foward is 37bp Tm:69.84,reverse is 40bp Tm:71.33 pcr product is 770bp,when I use this reaction pocedure I got a band but extremely unclear.94℃ 4min,1 cycle,94℃ 30sec,60℃ 10sec,72℃ 1min,30cycles,72℃ 10min,1 cycle,4℃ hold. How can I get more pcr product?Thank you!
do you need such long primers?
jane-00 on Thu Oct 14 08:13:58 2010 said:
I am cloning a gene fragment by pcr.But I cannot get pcr product.the primers:Foward is 37bp Tm:69.84,reverse is 40bp Tm:71.33 pcr product is 770bp,when I use this reaction pocedure I got a band but extremely unclear.94℃ 4min,1 cycle,94℃ 30sec,60℃ 10sec,72℃ 1min,30cycles,72℃ 10min,1 cycle,4℃ hold. How can I get more pcr product?Thank you!
Yes, why are your primers so long? and you have tried a gradient and failed, right?
gt_ameya on Thu Oct 14 09:06:12 2010 said:
jane-00 on Thu Oct 14 08:13:58 2010 said:
I am cloning a gene fragment by pcr.But I cannot get pcr product.the primers:Foward is 37bp Tm:69.84,reverse is 40bp Tm:71.33 pcr product is 770bp,when I use this reaction pocedure I got a band but extremely unclear.94℃ 4min,1 cycle,94℃ 30sec,60℃ 10sec,72℃ 1min,30cycles,72℃ 10min,1 cycle,4℃ hold. How can I get more pcr product?Thank you!
Yes, why are your primers so long? and you have tried a gradient and failed, right?
yes,I use the software "oligo 6" desgined a pair of primers,and get 29mer and 30mer two primer,and add restriction site and protecting base 10,at last I get a pair of long primers.I am a fresh hand,so......
Try a gradient then. Run the reaction at temperatures, 62, 63, 64 , 65 and 66 to see which one gives you a good yield. You might then have to optimise your reaction depending on your template DNA.
Good luck.... 
Thanks you,gt_ameya.I will have a try. but If I failed,what shoud I do. redesign the primers or change reaction conditions?
gt_ameya on Fri Oct 15 06:57:30 2010 said:
Try a gradient then. Run the reaction at temperatures, 62, 63, 64 , 65 and 66 to see which one gives you a good yield. You might then have to optimise your reaction depending on your template DNA.
Good luck....
Thank you,gt_ameya.I will have a try. but If I failed,what shoud I do. redesign the primers or change reaction conditions?
laurequillo on Thu Oct 14 08:50:09 2010 said:
do you need such long primers?
Thank you for your help!
How can I design a shorter primer? Can you give a guide to me? my pcr product is 770bp,28 mer of the primers complemet with the template DNA.I use the oligo 6. If you were I, what should you do?
jane-00 on Fri Oct 15 07:27:35 2010 said:
laurequillo on Thu Oct 14 08:50:09 2010 said:
do you need such long primers?
Thank you for your help!
How can I design a shorter primer? Can you give a guide to me? my pcr product is 770bp,28 mer of the primers complemet with the template DNA.I use the oligo 6. If you were I, what should you do?
You know you should calculate the Tm of the primers using only the "matching" sequence, right? you should not count all the new bases that you add.
What kind of template are you using in your pcr? We usually design primers with a lenght of 20 bps for genomic DNA. Adding a restriction site plus some additional bases at the 5`-end (for efficient cutting of the pcr product), you will end up at approximately 30 bps. But as laurequillo already stated, only the matching part of your primer are used for Tm calculation. You could also try to do more than 30 cycles in your pcr. But this also augments the danger of misincorporations.