again ligation problems
libra at ion.ac.cn
Wed May 15 07:15:35 EST 2002
You have done too much gel purification. I don't think that's necessary.
Institute of Neuroscience
Chinese Academy of Sciences
gianluca <gianluca.molla at uninsubria.it> wrote in message news:<gj52eucbmu5ckkcsbgfp4ian5s9mqh6msb at 4ax.com>...
> On 14 May 2002 14:09:30 +0100, mlsulliv at facstaff.wisc.edu ("Michael L.
> Sullivan") wrote:
> >Perhaps you could give a few more details about how you are preparing
> >the inserts.
> Sorry, I left some information in order not to be too long.
> So: usually I digest the DNA, load it in the gel, cut the bands
> (vector and/or insert), purify from agarose with macherey-nagel
> kit and then ligate.
> The PCR insert is prepared in different ways:
> - clean up the PCR reaction, (macherey nagel kit), then digestion,
> agarose gel separation, recovering of the band and again purification
> from agarose (MN kit).
> -load PCR products on agarose gel, purify the band (MN kit), digest,
> another purification (with or without loading digestion products on
> gel) and then ligation.
> As you see I tried several procedures.
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