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Molecular Cloning Forum DNA cloning forum. Discuss the cloning of plasmids, vectors, DNA, RNA, cDNA, proteins, and other molecules for expression, sub-cloning, and other methods. Also discuss digestion, ligation, transformation, plasmid preparation, cDNA library screening, and competent cells here.


Digestion problems

Digestion problems - Molecular Cloning Forum

Digestion problems - DNA cloning forum. Discuss the cloning of plasmids, vectors, DNA, RNA, cDNA, proteins, and other molecules for expression, sub-cloning, and other methods. Also discuss digestion, ligation, transformation, plasmid preparation, cDNA library screening, and competent cells here.


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  #1  
Old 02-16-2011, 10:45 PM
Pipette Filler
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Cool Digestion problems



Hi!

I have to do a double digestions to get my insert into the plasmid. The reaction buffers are incompatible to each other; 50-75% enzymatic activity. So, I'm doing separate digestion with PCR purifying kit after each digestion. But I never get the desired ligation product, only self-ligation, meaning one of the digestion was not done. My ligation reaction mix is correct.

So, is there any other way to do this digestion? Any help is appreciated.
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Old 02-16-2011, 11:15 PM
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Default Re: Digestion problems

Maybe one of your restriction enzymes is not working well due to salts or its just old/degraded.

Try to switch the digestion to the other enzyme first, and then make sure you check the results on a gel.

Maybe have to go the other way around
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Old 02-22-2011, 01:08 PM
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Default Re: Digestion problems

Maybe try to check the enzymes alone- so run a three lane gel with undigested, digested with enzyme 1 and digested with enzyme 2 to see if the vector becomes linear. Alkaline phosphatase can be used to prevent vector self-ligation.

If your insert is a PCR product (not changing vector) it might be worth checking the manual on the system that your products are 5'phosphorylated. A few systems require this for religation.
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