Subcloning large PCR fragments for transformations!

Ashok Mudgapalli ashok at LAMAR.COLOSTATE.EDU
Wed Mar 27 17:58:48 EST 1996


Dear Netters
I have amplified a PCR fragment of about 11.5 kb from D.melanogaster 
genome using Boehringer Mannheim's Expand Long Template PCR kit. I tried 
to subclone this fragment in to casper transformation vector and in other 
vectors with no success.
	Basically the PCR product has 3'-A over hang and I tried to 
polish the ends with Klenow or T4 DNA polymerase and tried to do blunt end 
ligation in to the casper vector. It did not give any positive clone. I 
also tried Stratagene's pCR script ligation kit. Here I have size 
limitation, the vector provided by the Stratagene can take fragments 
upto 10 kb. I tried this vector as a shuttle vector. This procedure also 
did not work. I have been subcloning various fragments (up to 15 kb) in 
to the casper vector without any problem, of course they are not PCR 
products.
	I would greatly appreciate if some could provide me few hints or 
their experiences in subcloning large PCR fragments in to the casper 
vector. 

All commercially available T-vectors do not have the capacity to hold 
such a large fragments (that was the information I got from different 
vendors).

Thanks in advance to every netter


Dr.Ashok Mudgapalli  



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