mhari at expert.cc.purdue.edu
Mon May 21 18:17:03 EST 2001
Howsoever I hate it, but I have to admit that I am unable to subclone a PCR product into my vector. Here are the specs :
PCR 1.5 Kb insert from a plasmid using Taq (RE sites internal to the primers so no question about sites missing)
Purify using QIAGEN PCR purification kit
Digest with HindIII and NotI(or SpeI)
Heat denature enzymes and ppt. DNA (tried both gel purification or without)
Vector digested with HindIII and NotI (or XbaI)
Run on gel to remove an insert already in the original vector (also confirms digestion with both enzymes)
Agarose band spun in a Sigma spin column and DNA ppted.
Checked purity and yield of vecotr and insert by running on gel and ligation setup with excess insert over vecotr at 15C for 24 hrs.
Control ligations include vector with ligase and no insert
RESULT : NO CLONES
I hope u have some ideas :)
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