Large DNA fragmnet ampification - (Aug/14/2008 )
I need to amplify a LARGe (12kb) GC rich fragment out of genomic DNA. Anyone has any good tips ? It would be great hepl!!
You must be patient. It is not a easy task.
The DNA must be of high quality. I advise you the kit Roche: Expand Long Template PCR System. But I can not say much more, I am starting with this technique. Good Luck
Hello,
I am also trying to amplify a 10kb fragment from genomic DNA. Since last 2 months. But no success. I have my primers designed with NotI and XhoI restriction sites at the end of fwd and rew primers. But what I get after ampification are multiple bands, instead of a single band. I did check for hair pin formation in the primers, but there were none. Any help will be really appreciated.
Thanks.
I am also trying to amplify a 10kb fragment from genomic DNA. Since last 2 months. But no success. I have my primers designed with NotI and XhoI restriction sites at the end of fwd and rew primers. But what I get after ampification are multiple bands, instead of a single band. I did check for hair pin formation in the primers, but there were none. Any help will be really appreciated.
Thanks.
catarino is right- first you need long dna fragments in your dna preparation - alqways check it on a gel. if you have high MW dna go on... i can recommend you the Expand Long Range dNTPack also from roche. it is even better than the Expand Long Template PCR System Kit and cheaper because it comes along with the dNTPs
sometimes increasing the annealing temp. or a touch-down pcr helps to increase specificity.
otherwise cut the right fragment out of your gel, repurify and re-pcr it than you have your pcr fragment.
Good quality DNA is a must.
You could also try Phusion for the long range PCR. Also look at the sequence that you are amplifying, try to avoid regions that have large arrays of short repeats.
And if worst comes to worst, see you can find a unique restriction site in your sequence so that you can amplify your sequence in two segments or more.
Remember to keep your eye on the goal, amplfication of the sequence and not how you do it. If you can't get as a single PCR product, see if you get it in segments that you can later rejoin. You can also use PCR to join segments of amplified sequences together. (if the two segments have a sequence overlap).
Hard task but if you will use polymerase for GC rich region it should work. This polymerase is on the market accesible
I am also trying to amplify a 10kb fragment from genomic DNA. Since last 2 months. But no success. I have my primers designed with NotI and XhoI restriction sites at the end of fwd and rew primers. But what I get after ampification are multiple bands, instead of a single band. I did check for hair pin formation in the primers, but there were none. Any help will be really appreciated.
Thanks.
catarino is right- first you need long dna fragments in your dna preparation - alqways check it on a gel. if you have high MW dna go on... i can recommend you the Expand Long Range dNTPack also from roche. it is even better than the Expand Long Template PCR System Kit and cheaper because it comes along with the dNTPs
sometimes increasing the annealing temp. or a touch-down pcr helps to increase specificity.
otherwise cut the right fragment out of your gel, repurify and re-pcr it than you have your pcr fragment.
Thanks,
I will try to increase the annealing temp from 60 to 65.
And if that works, I will also try the step you suggested, for isolating the band of interest and re-pcr it.