Troubleshooting ligation/transformation - (Nov/27/2005 )

Hi,

I'm trying to insert a 1.5kb fragment and a 2.5kb fragment (seperately) into pACYC184 - a low-copy E. coli vector with chloramphenicol resistance. The uncut vector is 4.2kb, and I plan to cut it with two enzymes - BglI and ClaI... ClaI cuts only once and BglI cuts twice giving total 3 bands: 3.1kb, ~900bp and ~230bp. Then I PCR my inserts with BglI and ClaI compatible ends and finally ligate to 3.1kb fragment.

Now the problem... the vector digestion seems to be fine (its a sequential double digestion) and I get the 3.1kb band (checked on gel). I gel purify the digested vector and insert, ligate and electroporate into E. coli DH10B cells. I got 50-100 colonies in the Cm plates... but when I do plasmid preps on those, I get plasmids of different sizes (ranging from 1.5kb to ~3kb)!! I should be ideally getting plasmids bigger than the starting vector (so >4.2kb).

I usually try to confirm presence of insert by PCR amplifying using the sequencing primers... when I do it for this case, I do get products, but again different colonies give different sizes (some of which seem to be the right size I would expect as if the insert is there).

Then I tried to do restriction digest on those that seem to have insert according to PCR results with two enzymes so as to get different gel patterns... Both pACYC184 and the plasmids I generate after this cloning should give two bands but different sizes in each case, but I see only one band for the plasmids I generate. The base vector pACYC184 digestion works fine giving two bands of correct size.

I'm confused now as to why my plasmids have different sizes from different colonies and are smaller than the starting vector when I should get a bigger one? Till digestion it seems to go fine, but after that I get all wierd things Any input will be appreciated.

-Pachak

Hi,

I'm trying to insert a 1.5kb fragment and a 2.5kb fragment (seperately) into pACYC184 - a low-copy E. coli vector with chloramphenicol resistance. The uncut vector is 4.2kb, and I plan to cut it with two enzymes - BglI and ClaI... ClaI cuts only once and BglI cuts twice giving total 3 bands: 3.1kb, ~900bp and ~230bp. Then I PCR my inserts with BglI and ClaI compatible ends and finally ligate to 3.1kb fragment.

Now the problem... the vector digestion seems to be fine (its a sequential double digestion) and I get the 3.1kb band (checked on gel). I gel purify the digested vector and insert, ligate and electroporate into E. coli DH10B cells. I got 50-100 colonies in the Cm plates... but when I do plasmid preps on those, I get plasmids of different sizes (ranging from 1.5kb to ~3kb)!! I should be ideally getting plasmids bigger than the starting vector (so >4.2kb).

I usually try to confirm presence of insert by PCR amplifying using the sequencing primers... when I do it for this case, I do get products, but again different colonies give different sizes (some of which seem to be the right size I would expect as if the insert is there).

Then I tried to do restriction digest on those that seem to have insert according to PCR results with two enzymes so as to get different gel patterns... Both pACYC184 and the plasmids I generate after this cloning should give two bands but different sizes in each case, but I see only one band for the plasmids I generate. The base vector pACYC184 digestion works fine giving two bands of correct size.

I'm confused now as to why my plasmids have different sizes from different colonies and are smaller than the starting vector when I should get a bigger one? Till digestion it seems to go fine, but after that I get all wierd things Any input will be appreciated.

-Pachak