Cloning question

huangz huangz at usa.net
Fri Apr 9 08:38:46 EST 1999


Dear bionetters,

I have a 3.8 kb PCR product which amplified from rat genomic DNA is waiting
for sequencing. I want to clone it with either of the following strategies
and hope someone can give me valuable advise on that.

method 1.I can T/A cloning the 3.8 kb PCR product with a T vector. But as I
know the T vector is suitable for a fragment less than 700 bp and the
transfection efficient will be dramatically decrease if such a long (3.8kb)
product is inserted. I was also heard that invitrogen supply a new TA vector
which can handle 3-10kb PCR product, but I am wondering if anyone has a
successful experience with this vector.

method 2. I have to design new primers which includes MCS enzyme site (It
can be done with certain mutation in the 5'/middle part of the primer). And
amplify again with the new primers. The PCR product will then be cutted with
appropriate enzymes (BamH I and Hind III). A universal cloning plasmid
vector (such as pBluescript II SK plus or PCR II) will be treated with the
HindIII and BamHI thus directional cloning can be performed subsequently.

I am wondering which method is suitable for my task. Any comments or even
better solution are greatly appreciated.

Dr.Han
Biogate





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