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Screening for ligation and transformation result


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#1 DrLeo

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Posted 27 March 2013 - 09:56 PM

Hi all,
After ligation of insert and pGL3 vector, we will transform the vector w/wo insert into CP cells.Thus, how could you choose the colony which contains the pGL3 including the insert?
PCR and enzyme cutting screening, which is better?
Thank you for your help.

#2 GNANA

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Posted 27 March 2013 - 10:31 PM

i dnt think there is a big difference between these two methods provided if you have a proper controls, however, restriction digestion is easier and to some extent you can avoid false positives/negatives compared to PCR. so always my first choice would be enzyme digestion........

good luck..
I would prefer being perfectionist rather than a passionist in Research.

I always had an alternate hypothesis....

#3 HOYAJM

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Posted 28 March 2013 - 10:06 AM

I go with miniprep + sequencing. If you eventually have to sequence a construct, I figure I might as well do it at the beginning. I usually pick 6 colonies and send them for sequencing.

I used to screen everything by colony PCR, and false positives can be a problem. To me, I choose to do RE digests last, its just time consuming.




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