xhoI cloning problems

suter at de.d400.mpg.MPIZ-KOELN.VAX suter at de.d400.mpg.MPIZ-KOELN.VAX
Thu Sep 23 10:30:12 EST 1993


as written by: S=amcshea;OU=world;O=std;P=com;A=d400;C=de 
in answer to a question of mine about cloning with XhoI:

+......
+I have also cloned with Xho-1 till I was blue in the face trying to get
+genes into retroviral expression vectors.
+	Has anyone tried the Pae R7I enzyme from Biolabs which is an
+isoschizo of Xho-1 and pretty cheap, if wonder if this is such a pain in
+the arse, or whether it works.
+	Good to hear we are not alone.....!
+		Happy cloning,	Andy.

I seem to have solved the problem with XhoI finally. Basically, i just mixed
bluescript and lambda DNa in one vial, digested with XhoI, heat inactivated
20' at 65C and ligated o/n at 16C. analyis on a gel reveiled that ligation
had taken place. remember, many different trials preceeded this desperate 
experiment !
transfection of e.coli resulted (naturally) in 10.000's
of colonies, of which four lighted up after turbo screening. I hope
these are true positives.

   I compared my reaction conditions to find an optimum:
1. dna was present during ligation at 200ug/ml (less or more looked worse),
equal weights from lambda and pBS 
2. xhoi was from biolabs, ligase and buffer from BRL (others didn't work)
3. sap and cip were impossible to use, several tries
4. proteinase K and phenol to purify insert and/or vector did not help (was
aborted however, after positives were obtained).

all in all: i am glad to hear there exists an isoschizomer for xhoi. i just
wasted 3 weeks for a 'standard' subcloning experiment. 
thanx for all the advice.

cheers, clemens
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Dr. Clemens Suter-Crazzolara, PhD
Max-Planck-Institut fuer Zuechtungsforschung
Abteilung Genetische Grundlagen der Zuechtungsforschung
Carl-von-Linne Weg 10,        D-50829 Koeln, Germany
Tel.xx49.221.5062-221    Fax.-213      e-mail: suter at vax.mpiz-koeln.mpg.d400.de
===============================================================================
	if this is victory, our hands are to small to hold it
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