cloning PCR products etc

Jonathan Moore jdmoore at galactose.mc.duke.edu
Fri Jan 20 15:04:06 EST 1995


Dear list,
	a couple of questions. What is the best way to subclone PCR 
amplified fragments into expression vectors?

	Currently I amplify from the template, chlororofm extract to get 
rid of the oil, then I perform 1 PCA and 1 chloroform extraction and 
ethanol precipitate. The pellet is resuspended then digested with the 
enzymes and then run on a low melt gel. I purify the DNA I cut out by the 
freeze sqeeze method, PCA and chloroform extrcating and ethanol 
precipitating before putting it into the ligation.
	Theres about 15ng of cut (and phosphatased) vector in the ligation.
	Result - very few colonies.

Question: Does anyone share my suspicion that phosphatase treating 
reduces the ligation efficiency (I am treating the cut vector with PCA etc)

Question: Would it be better to digest the PCR product AFTER purifying it 
from a gel rather than BEFORE.

Question: Does anyone think my problem is agarose derived impurities 
affecting the efficiency of the ligation

Any replies/suggestions will be appreciated. Would commercial 
silica/glass bead based DNA binding resins help here at all???

Please reply to me by e-mail:

	jdmoore at galactose.mc.duke.edu

Thanks in advance 



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