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A Question about purification in making clones


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#1 popogirlxd

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Posted 05 January 2005 - 10:01 PM

Hi,everyone
Could any one tell me how many times gel purification I should perform in making clones. For EXample, I am doing a blunt-end ligation. I cut my insert from a plasmid with differet REs,and blunt the end with klneow. As to the vector ,I first cut it ,then blut the end and dephosphorylate the end. After this, I ligate the vector and insert together. So during the procedure, should I do gel purification after each step before next step?? You know, I found gel purification make lot of insert and vector lost which makes me feel upset.
Could you give me any suggestion? Thank you!!!

#2 leahf

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Posted 08 January 2005 - 11:29 AM

After every step you can try cleaning the vector by spin-collumn purification (like a PCR kit, if vector is not too large) followed by (if necessary to increase concentration) ethanol precipitation.
But -
the first step of preparing your vector (the restriction) it is usually a good idea to purify the cut form from gel, because if you have even a small amount of uncut vector left over, this amount could give you a whole lot of background when doing the transformation.
you can test for this by a doing a control transformation with your cut vector (without gel purification) - if you get very few transformants, it should be o.k.

#3 popogirlxd

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Posted 09 January 2005 - 06:46 PM

Thank you for your advice. I will definitely take it!




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