subcloning - (Jul/06/2005 )
Hi,
I am trying to subclone a number of inserts ranging from 207 bp to 726 bp into a 6008 bp vector using EcoRI and BsrGI (double digestion using NEB buffer 2 + BSA) . These two enzymes are 60 bp apart in the vector (Kanamycin resistance).
So far, I havn't been able to get any colonies on my plates.
I have checked all the following steps:
1. I know both enzymes are cutting the vector because I cut using each one individually alongside the undigested plasmid.
2. After gel extraction of insert and vector, I ran a small amount (~50 ng) on a gel to make sure I didn't lose my DNA before ligation.
3. The plates (30 ug/mL Kanamycine) are freshly prepared and the compentency of my cells is fine.
4. I have tried using CIP to reduce vector religation, and I also tried without using CIP. Both times, I didn't get any colonies, even on the plate with just the religated vector.
I cannot understand what mistake I am making. Any suggestions would be really appreciated.
Thanks!
Roshni
Everything sound good near as I can tell.
Bad ligase? Wrong temp for ligase? Did you inactive your ligation rxn prior to transformation?
HI Roshni29,
As far as I can tell it is definetely not your technique. The only thing I can think of is if, as mentioned, the ligase/ligase buffer is OK. I would grab a fresh aliquot (completely untouched) of both out of the freezer, or potentially add ATP to the ligase buffer (it goes off reasonably rapidly, especially with freeze/thaws).
pBluescript do you always inactivate your ligase before transformation? I tend not but might improve my efficiency on difficult ligations, might give it a go next time I have problems.
Cheers,
Scott
Yup, 70C 10 min. Quench on ice, quick spin and then add to cells.
As far as I can tell it is definetely not your technique. The only thing I can think of is if, as mentioned, the ligase/ligase buffer is OK. I would grab a fresh aliquot (completely untouched) of both out of the freezer, or potentially add ATP to the ligase buffer (it goes off reasonably rapidly, especially with freeze/thaws).
pBluescript do you always inactivate your ligase before transformation? I tend not but might improve my efficiency on difficult ligations, might give it a go next time I have problems.
Cheers,
Scott
Hi,
I got a couple of inserts to ligate but still having trouble with the rest. This means that the effiency of either the digestion, or the ligation is very low. I tried digesting the vector first with BsrGI and then with EcoRI. I gel purified the vector. But, I still got nothing on my plates. I really don't know what to try next!
I always inactivate the ligase before transformation. I tried using commercial competent cells. I made new plates.
Please help!
Roshni
May I know How do you prepare your insert? Was your insert made from PCR product?
If so, please check the protective bases that besides the restriction site on your primer, sometimes the protective bases make restriction enzyme doesnt work.
Hi,
I am also interested in bullfrogs question, if you are cloning pcr products, perhaps it will help to subclone into a t-easy vector or equivalent before doing the digestion for ligation. This can help in several ways, the enzymes seem to cut better, and you can get more insert from a plasmid prep than from a PCR.
I have also used the following protocol for in-gel ligations when I have had particularly difficult clones to obtain. It is a quick and easy protocol, and has worked for me at times when I was having similar problems to yours. I can't explain why it worked when gel-extraction did not, but it really made a difference in my hands.
www.flemingtonlab.com/Protocols/LowMeltLigationProt.pdf
Hope this helps, and Good Luck!!
My insert is orginally prepared by PCR, then blunt cloned into another plasmid. I did a miniprep of this plasmid, and cut with EcoRI and BsrGI. I see a very clear band of the right size after digestion which I extract using Qiagen gel extraction kit.