Cloning a long PCR product - (Aug/20/2004 )
I've done a RACE PCR from an EST (with Taq) and I have a product which is about 6kb long.
Since it's a RACE product, I don't know what it is and I wanted to clone it into a vector to get it sequenced.
So, I've tried to clone it by using the TOPO TA Cloning kit for sequencing (from Invitrogen, the vector provided in this kit is pCR 4-TOPO vector, 3954bp long). But it didn't work. I've done all the controls I could at the same time and they were fine.
The problem is I have very tiny amounts of this product and I don't want to loose it. That's why I'm looking for some good advice.
I know one thing I could do is to try different ratios (I had chosen a vector:insert ration of 3:1) but this is gonna make me using a lot of my product.
Are there vectors available that are especially suitable for such large PCR products ?
What would you advice me to do ?
Thanks for your answers.
maybe this helps: http://home.uchicago.edu/~bchen2/PCRCloning.htm