Emergent-----Cloning problem

Haojiang Luanhaojiang at hotmail.com
Fri Nov 22 13:26:28 EST 2002


Thank you for the response. What I did is 1. Use pBSK+ to insert the
first clone fragment(three KB, all inserted fragment will be from
mouse). Then destroy the NotI site by NotI digestion, blunt it and
religate. Then after Xho I digestion, I am trying to insert the second
XhoI fragment (4.7KB). The insert were purified by Qiagen Gel
purification kit(then by QiaQuick PCR purification kit, because the
one the one purified by Qiagen Gel purification kit didn't work very
well). The vector (6.2KB) was dephosphaylated by CIP then purify with
QiaQuick PCR purification kit. I did the ligation with Roche quick
ligation kit and the common NEB ligase(overnit ligation). Then
transform into Stable two competent cells. After use use probe to find
the colonies that may have the insert, we make miniprep of those
colonies. But they are just the same size as vector. But I didn'y try 
to recover the insert form these construct. I will try.

Haojiang



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