21 replies to this topic

[jane-00]

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!

[laurequillo]

do you need such long primers?

[gt_ameya]

jane-00, on 14 October 2010 - 12:13 AM, 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?

[jane-00]

gt_ameya, on 14 October 2010 - 01:06 AM, said:

jane-00, on 14 October 2010 - 12:13 AM, 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......

[gt_ameya]

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....

[jane-00]

Thanks you,gt_ameya.I will have a try. but If I failed,what shoud I do. redesign the primers or change reaction conditions?

[jane-00]

gt_ameya, on 14 October 2010 - 10:57 PM, 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?

[jane-00]

laurequillo, on 14 October 2010 - 12:50 AM, 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?

[laurequillo]

jane-00, on 14 October 2010 - 11:27 PM, said:

laurequillo, on 14 October 2010 - 12:50 AM, 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.

[ElHo]

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.

[phage434]

Step 1: try your pcr with an annealing temperature of 55C.

[jane-00]

ElHo, on 15 October 2010 - 02:08 AM, said:

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.

thank you！I will have a try！

[leelee]

laurequillo has a good point, you should only use the 28mer that is complementary to your template to calculate the Tm. I think you will find this drops the value considerably.

I would do as phage434 and run your PCR with a 55C annealing and see what happens. You could even run your gradient starting at this temp or there abouts.

[jane-00]

phage434, on 15 October 2010 - 03:53 AM, said:

Step 1: try your pcr with an annealing temperature of 55C.

Thank you,I have already pcr at this temperature but it didn't work.

[Adrian K]

jane-00, on 17 October 2010 - 12:37 AM, said:

phage434, on 15 October 2010 - 03:53 AM, said:

Step 1: try your pcr with an annealing temperature of 55C.

Thank you,I have already pcr at this temperature but it didn't work.

What you mean by it didn't work? at 55C you see no bands, but you mentioned previously at 60+ there are faint bands?

You can try a long range gradient 55C to 65C.
Also, what is the calculated annealing temperature for each of your primers? Only the matching site without the RE added sequences?

Or, try to excise the faint band and do a pcr on it?

Edited by adrian kohsf, 17 October 2010 - 08:17 AM.