Cloning of high-GC% gene into vector
Posted 09 April 2003 - 03:49 AM
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?
Posted 25 April 2003 - 04:40 AM
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 !
Posted 25 April 2003 - 07:03 AM
Posted 29 September 2003 - 05:34 AM
The kit is not expensive about 40$/25reaction. So you can try it.