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re-digestion - (Mar/18/2010 )

Hi everyone

I am trying to do cloning.
I double digested my PCR products and vector (which had another insert). I have seen that this other insert was cut, but the band was not very strong.
I made the ligation and transformation so in the end I had very nice colonies-I thought. However today I got the sequences and seen that all the colonies were negative with respect to what I tried to insert and instead positive wrt the insert I thought I cut out.


So I think now my restriction was incomplete and I will repeat it now. I already have some of my digested PCR product (the one I try to insert). My question is, can I digest it again? Or should I make the PCR again and digest that?


Additional info: I have used NheI and EcoRI enzymes, digested in NEBuffer 1 + BSA at 37C for 2 hours. Should I increase the time?


Thanks in advance!

a poor, newbie master student :D

-cellgene-

cellgene on Mar 18 2010, 04:44 PM said:

Hi everyone

I am trying to do cloning.
I double digested my PCR products and vector (which had another insert). I have seen that this other insert was cut, but the band was not very strong.
I made the ligation and transformation so in the end I had very nice colonies-I thought. However today I got the sequences and seen that all the colonies were negative with respect to what I tried to insert and instead positive wrt the insert I thought I cut out.


So I think now my restriction was incomplete and I will repeat it now. I already have some of my digested PCR product (the one I try to insert). My question is, can I digest it again? Or should I make the PCR again and digest that?


Additional info: I have used NheI and EcoRI enzymes, digested in NEBuffer 1 + BSA at 37C for 2 hours. Should I increase the time?



Thanks in advance!

a poor, newbie master student :D





You could digest it again. But, in such cases I always like to start afresh, ie from the PCR. Also, for preparing my insert/vector for ligation purposes, I use longer time for incubation 4-5 hrs.

Best

-DRN-