Cloning PCR products

Geoffrey Kidd GKidd at Aptagen.com
Mon Oct 27 12:42:34 EST 1997


One factor to watch out for is U.V. exposure.  A large plasmid such as 
yours makes a larger target for U.V. damage than do the 2-3 kb 
varieties.  Be sure to block as much U.V. as possible when excising from 
the gel, allowing just enough fluorescence to see your DNA, and excise 
it as quickly as possible.  I found that our light box was able to 
destroy all of a 3 kb plasmid after about 5-6 minutes. 

As a control to see if this is a problem, just try re-ligating some cut 
plasmid, without insert, before and after gel-purification.

Geoffrey Kidd

Matthew Knight wrote:
> 
> Hi all,
> 
> I am currently trying to clone a 0.65 PCR prod into a 7.2kb vector
> with great difficulty. I am using two different enzymes and have tried
> several different ligation ratios of 1:1, 3 insert: 1 vector and 5 insert
> : 1 vector.
> 
> I am digesting my PCR product and I am sure it is cutting due to a
> size differnece on agarose gel and then I am heat inactivating and ethanol
> precipitating the DNA.
> 
> I am gel purifying my vector with a silica matrix. My control
> transformation is working fine into E.coli TG2.
> 
> Does anyone have any suggestions on ligation ratios and DNA
> concentrations. For example I am ligating approximately 30-50ng vector
> with between 5-15ng insert.
> 
> Thanks in advance.
> 
> Cheers
> Matthew Knight
> Centre for Bioprocessing and Food Technology
> Victoria University
> Phone 61 3 9216 8137
> Fax   61 3 9216 8135



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