dspinella at chugaibio.com
Thu Jun 18 17:32:23 EST 1998
> We have been trying to clone cDNA into either the T7 Select vector or
> Stratagene's Lambda Zap vector. Regardless of the cDNA or the
> adapters that we use, we consistantly only clone adapters and no
> cDNA. We have used several types of column chromatography and NH4OAC
> precipitations to try and clean the free adapters from our cDNA.
> Even when we use our column chromatography and select fractions
> that should contain only large DNA (>800bp), we will still only get
> free adapters cloning. Has anyone else experienced this problem and
> if so....how do you effectively get rid of all of those adapters?
I've always used a 1% agarose gel to size-select cDNA. Column
chromatography (as you observed) just doesn't have the resolution to
completely remove the adaptors -- and (also as you observed) they clone
with very high efficiency. Just isolate the cDNA smear above ~600 bp.
This has always worked great for me. Good luck.
PS You might also consider non-palindromic BstX1 adaptors.
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