3 replies to this topic

[boericyu]

Hi,

I am trying to ligate a high-GC% gene (~3.7kb)  into pEGFP series from BD Biosciences, but it's hard to be cloned! I am wondering what condition I could try?

[warp]

Hi Boericyu,
I guess that you encountered some problem with you PCR reaction...
For GC rich templates I'm used to add some DMSO (know to help with GC rich) and to higher a bit the denaturation temperature (aslo know to help). With those 2 improvement, i usually get trough difficult GC rich sections.
Now, you have to do a couple of optimizations ......
To ease that, I am using a very thermostable enzyme that has DMSO in its runing buffer (isis pol from qbiogene). It is an hifi dna pol, but I just don't care about that ...
it is easy to use, and save me time during my optimization.

Have fun !
WARP

[bouttela]

I had a similar problem and fixed it by using Thermalace by Invitrogen...it was much easier than fooling around with formamide and DMSO.  This pol is awesome.
Good luck

[lzfly]

I also work with GC% rich template, I chosen TaKaRa LA Taq with GC Buffer to amplify and easily cloned 430bp, 2.2kb, 4.3kb fragments (GC  70%).
The kit is not expensive  about 40$/25reaction. So you can try it.
Best wishes!