Yes, is not that easy, but there are several long range polymerases available on the market which can amplify templates up to 20kb. But in some case it is a lot of work to get the pcr running...
Yes, is not that easy, but there are several long range polymerases available on the market which can amplify templates up to 20kb. But in some case it is a lot of work to get the pcr running...
I've done 6 kb with few problems in the past, and I've seen claims of up to 30 kb. But Florian is right: careful optimization is needed. Keep in mind that there can be template sequence-specific issues along with concerns about enzyme processivity and fidelity.
That depends on the sequence, some sequences are harder to amplify than others. I will most likely take some time and work to get there.
Trying to desing good primers and depending on your DNA sequence (content on GC), you could amplify w/o any problem. I do it twice during my PhD...
Yes, and it is not that difficult. We use GoTaqLongPCR Mastermix (Promega). And the optimization doesn't take that long. In most cases the time needed for extension was the critical point as we amplified up to 12 kb. But the specifity of the primer pairs is a prerequisite to get a distinct band on the gel.
Hi Anjan, Yes it is possible amplify 10kb sequences. I used to use a mixture of Taq and Pfu polymerases. Some of the commercial products are such blends. I have used this mix successfully to 11kb amplifications. I used 1:1 mix.
Alternatively, if your fragment is in a plasmid, you can use recombineering-mediated gap-repair. In that case, you do not need to sequence the entire fragment.
I would give the Q5 polymerase from NEB a try, it worked very well for me for a 5kb amplification from genomic DNA.
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