Ligation, plasmid rescue problem! - PLasmid transformation after ligation problem (Jan/02/2008 )
Dear All;
I have been having a problem since 2 months. I am ligating a 2.7 kb inser and a 3.4 kb insert into a 8 kb vector. Enzymes ends: KpnI, EcoRV and XhoI. I used negative control for ligation (where I only ligate my cut vector) and it showed no colonies. All plates of ligation reaction (after transformation) show colonies and seem great. However, when I do the miniprep, and run the gel to confirm the plasmid, I get different plasmid sizes all over. Sometimes Ig et just s a ~3KB fragment sometimes I get lower but never 14Kb. Thus the bacteria is growing in Amp and it looks like ligation is working (although I have no positive controL) but I dont get my desired plasmid.
I think it could be recombination problem. That is the ligation is working but plasmid is recombining in bacterial genome and then some parts just leave the genomic DNA. So, I tried growing it at lower temperature (32 or 30 instead of 37) and I tried lower shaking speed... I used DH5a and sbtl2 strains yet same results.
Anyone has any suggestion?
Please reply.
Thank you
could you confirm what the insert digestion for this 3 way ligation?
Is it
Kpn1---Insert1---Xho1
Xho1---Insert2---EcoRV
Kpn1---Vector---EcoRV
Next how many colonies are you screening? And by the way what kind of check digest did you conduct?
I would suggest that you conduct colony PCR (using a PCR reaction that amplifies across the junction between inserts) to sceen. As you are now well aware screening by miniprep is a very time consuming process. Wth colony PCR and a multichannel pipette you can screen 72 colonies easily.
Just to confirm matters, are the DNA fragments clean? After digestion, were the DNA fragments gel purified?
As you have suggested, one possibility is the recombination problem. I would try SURE cells to overcome. Also can you describe your ligation mixture.
Thanks for dropping a reply. Well I don't know about SURE cells, are these teh expensive fancy ones? It is not sold by invitrogen, is it?
Well i did different ligation mixtures but always the vector was low compared to inser... like at least 3 times.. and for once it was in equimolar...Does this answer yoru question?
Is it
Kpn1---Insert1---Xho1
Xho1---Insert2---EcoRV
Kpn1---Vector---EcoRV
Next how many colonies are you screening? And by the way what kind of check digest did you conduct?
I would suggest that you conduct colony PCR (using a PCR reaction that amplifies across the junction between inserts) to sceen. As you are now well aware screening by miniprep is a very time consuming process. Wth colony PCR and a multichannel pipette you can screen 72 colonies easily.
Just to confirm matters, are the DNA fragments clean? After digestion, were the DNA fragments gel purified?
I am sorry, I didn't see your reply earlier.
It is Kpn1-insert-EcoRV-insert-Xho1- Vector
I have screened ~ 200 colonies for now and you can never imagine how boring is that. I have done different ligations as well. The DNA is not so clean. I don't know why but whenever I gel extract anything using Qiagen, the 260/230 is too low (ethanol and salts). I tried washing mroe than once with PE yet the same. 260/280 is normal. The amount I am getting back from Qiagen columns is much less than what I used to (when I worked in another lab). I order a new kit yet the same is happening.
I am still having a problem if anyone has any suggestioN!
I am uploading an image of a gel I ran of the plasmid i miniprep.
Well i did different ligation mixtures but always the vector was low compared to inser... like at least 3 times.. and for once it was in equimolar...Does this answer yoru question?
Sure cells are available from stratagene.
For some difficult ligations, i would use 5 -10 x more insert (1:5 or 1:10 ratio of vector: insert) in the ligation.
In your ligation add ~100 ng of vector and insert in 1:5 ratio. This may help.
For gel extraction, try invitrogen. I used to like Qiagen but now have the same problem as you are facing, so switched to invitrogen.
good Luck !!!
Well i did different ligation mixtures but always the vector was low compared to inser... like at least 3 times.. and for once it was in equimolar...Does this answer yoru question?
Sure cells are available from stratagene.
For some difficult ligations, i would use 5 -10 x more insert (1:5 or 1:10 ratio of vector: insert) in the ligation.
In your ligation add ~100 ng of vector and insert in 1:5 ratio. This may help.
For gel extraction, try invitrogen. I used to like Qiagen but now have the same problem as you are facing, so switched to invitrogen.
good Luck !!!
Malik;
Do you think it is recombination problem? I tried DH10B Max efficiency and Sbtl2 cells still getting same result.
Do you think it is recombination problem? I tried DH10B Max efficiency and Sbtl2 cells still getting same result.
I think recombination could be a factor. Even if you try cells which prevent recombination, they do not completely prevent recombination. From what you have described, like having many colonies but of varying sizes, sounds like recombination. I have actually had problems with recombination and from what you described, it seems like that. I have used XL-gold and SURE cells from strategene for this problem. Other cells types might work too. Try increasing the total DNA in ligation and also have ratio of 1: 5 or 1:10 and give it a go. I solved by using very high conc of DNA in ligation like vector 150ng and insert ratio 1:5 and it worked for me. But think of other likely problems as well. Like over digestion of the ends.
Good Luck !!!