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Restriction double digest

Cloning

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#1 Ramiz Sheikh

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Posted 07 December 2014 - 04:31 AM

Hello everyone,

 

I want to subclone my gene into pet22b vector. I did double digestion of both vector and insert plasmid with Nde1 and HindIII 1ul each in 50ul reaction with buffer 2 and incubated for 3 hours at 37degree.  Also as controls i digested my samples with individual enzymes also. After agarose, i found out that the vector was linearized and the band was moved upward than the uncut vector. The problem is with my insert plasmid. With individual enzymes, i am getting a single band of linearized plasmid. However, with double digestion, 3 bands are seen which concludes that the plasmid has not been digested. Everytime i get different results with double digestion. i think enzymes are working fine.. Can any1 please shed some light on this?? Thank you



#2 phage434

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Posted 07 December 2014 - 06:47 AM

What is your setup for the restriction digest that is not working? (How much volume of each of the components)

A common problem is inhibition by impurities in the minipreps of plasmid DNA. This happens most often when the major volume of the digestion is dilute DNA solutions, rather than water.







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