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Do you have to purify target vector after double digest from MCS piece

MCS double digest purify ligation

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#1 assembler01

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Posted 11 January 2013 - 09:06 AM

I put a pcr fragment (with 2 primer introduced restriction sites) into pCR2.1-TOPO vector. I'm digesting that, and the pMir vector with 2 restriction enzymes. I'm gel purifying the insert (with SYBR & blue light as long as I can get them to work). Now, my question is, do I have to gel purify the pMir target vector since the small piece of the multiple cloning site will still be there to cause re-ligation or ligate to the insert. I will phosphatase treat the pMir target vector, but does that guarantee the MCS piece will never re-ligate and what about the insert-MCS piece?

We have a pcr/gel-purification column kit that I could try to use to purify the MCS piece from the vector but I don't know how effective that is. It claims it removes DNA fragments less than 100bp, but how efficient is it? Am I better off gel purifying it, or will that just potentially risk degrading it if I have to use UV light? Of course, if the restriction digest doesn't look complete, I should gel purify it anyway to get rid of uncut vector, but I'm optimizing the digestion reaction now. Are there any papers out there showing the percent transformants of MCS versus insert after a double digest?

Thanks.

Edited by assembler01, 11 January 2013 - 09:11 AM.


#2 HOYAJM

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Posted 11 January 2013 - 10:33 AM

You should go ahead and gel purify the vector. Those spin columns for DNA clean up can have low yield and in my opinion is not as good as gel purifying. Also, as you said, you can gauge whether or not the digestion was complete when you run it on the gel. It adds an extra 1-2 hours to the process but it will pay off when you dont have to screen 30 colonies to get a clone.





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