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 37°C for 2 hours. Should I increase the time?


Thanks in advance!

a poor, newbie master student

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 37°C for 2 hours. Should I increase the time?



Thanks in advance!

a poor, newbie master student