cloning and transformation - (Apr/24/2008 )

i cloned the insert in the vector of interest and transformed it into ecoli. i tried to confirm the insertion by colony PCR and i got poistive results, i.e. getting a pcr product of expected size etc. BUT, when i did minipreps and did a double digestion , i see no band at the size of the insert!, i tried confirming if i had the insert in theminiprep DNA, so i used my miniprep DNA as my template, this time also i got the PCR product as expected.

I thought maybe the double digestion is not working well in these minipreps. so i tried digesting with individual enzymes, and each of them cut well.

i am very perplexed. the enzymes i am using is Pac1(NEB) and BamH1(Promega).

Hoping a response.

hi,

have you made two sequential digestions or used the 2 enzymes in a unique buffer?
in this last case, which was the buffer? NEB4?
check this web site for double digestions:
http://www.protocol-online.org/forums/inde...6&hl=citric

Sebastien