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cytotrap library construction problem

Rory Koenen r.koenen at bioch.unimaas.nl
Fri Jan 21 10:44:05 EST 2000


Hi,
Make shure that the cDNA is free from residual polymerase activity (if you
do sticky end ligations), because the polymerase may fill up the 5'
overhang. I had this problem just a week ago (I was too lazy to clean my
insert properly). Multiple phenol extraction or silica/agarose gel
purification will do the trick.
Good luck,
Rory

Frederik Börnke wrote:

> Hi all,
>
> we are desperately trying to construct a cDNA library from tobacco
> for Stratagene's CytoTrap THS. However, although we yield plenty of
> cDNA (1.5 - 2 µg) ligation efficiency into the precut pMyr vector
> is very poor. We tried several ratios between vector and insert without
> any positiv effect. The controls for ligation and transformation
> are quiete persuasive. Thus, it seems that there is something wrong
> with our inserts, like inefficient linker ligation or else. Has
> anybody encountered trouble like this? Would an additional round
> of linker ligation help? Any hints will be appreciated.
> BTW, if anybody has a library prepared from plants and would be
> willing to share such a library, we would be very pleased.
>
> TIA
> Daniel and Ricky
>
> *******************************************************************
>
> Frederik Boernke
> Research Group of  Molecular Plant Physiology
> Institute for Plant Genetics and Crop Plant Research (IPK)
> Corrensstr. 3
> 06466 Gatersleben
> Tel.  039482 -5 321
> Fax. 039482 -5 515
> http://www.ipk-gatersleben.de





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