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Problems with ligation and transformation


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#1 zhangj3

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Posted 27 May 2011 - 05:29 AM

Dear all
I have a big problem with ligation and transformation reactions. I have basically 3 types of ligation reactions:
(1) Vector and insert are both cut with EcoRI, vector is CIPed.
(2) Vector and insert are cut with two different enzyme
(3) Vector is cut by one adhesion end and one blunt end, insert was blunted by T4 polymerase and then cut with a second enzyme
However, after ligation and transformation, only the first reaction gave me colonies. But there are equal numbers of colonies in both vector only vs vector plus insert. I miniprep 8 of them, they are all vector self-ligation. I tried 4 degree ligation vs room temp, it's the same result. It seems my ligase is OK, given the fact that vector self-ligation happens, maybe the CIP is not good. But I really have no clue why the other reaction never gave me colonies.
Many thanks
Ji

#2 Penny

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Posted 27 May 2011 - 09:03 PM

Hi, if you did not get any colonies. The highly possible reason is the vector and insert did not cut completely. how long did you digest your vector and insert? I was using fast digestion enzymes from invitrogen. Sometimes it will need more than 1 hour to digest the vector or insert completely. Now, I usually do the digestion for 4h or sometime overnight. It is alway good to check the digestion results on the gel before u go for the ligation. Make sure the single, clear band was achieved after the digestion. hope this helps

#3 zhangj3

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Posted 30 May 2011 - 11:29 AM

I always check the digestion efficiency by running a gel. Indeed, it's perfectly digested at least for the insert ( I can see two bands at differet size correctly). I am not sure whether that means the double digestion of the vector not work, since I can't judge based on the size of the band, it looks the same as sinlge digestion.




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