Subcloning problem with XhoI digested fragment - weird pattern of migration/digestion after miniprep (Jul/21/2006 )

Hi erveryody,

Yes, I have a problem.
Briefly, I'm trying to subclone a 5kb fragment from pCRII (4kb) plasmid in a 7.5 kb bgal expressing vector. I'm also trying to subclone a 3 kb fragment from pBS-KS (3kb) in the same bgal expressing vector. My restriction site is XhoI. After XhoI digestion, I purified my 5 and 3 kb fragment by gel extraction and got a clean band on my gel. I also cipped my vector. I did 24h ligation at 16°C, using multiples molar ratios (From 3:1 to 15:1 (insert:vector)). I transformed my ligation products either by heat shock in DH5alpha or by electroporation in Top10 competent cells. I have multiples colonies on my plate but just a few on my control plate (the digested and cipped vector, so the vector is well dephosphorylated). When I analyzed the colonies obtained I never get the correct ligation product (confirmed by xhoI digestion). Instead I have a strang pattern of migration, for the "3kb fragment + the bgal vector" I obtained a non digested band around 1.6k, and for the "5kb fragment + the bgal vector", I obtained a band at 3.5kb. I also tried to do the same thing with another bgal experssing vector and got the same weird results.
So I don't know where is the problem. Do you think that I can have contamination with my pCRII or my pBSKS vector even if the purified fragment look good?
Do you some ideas for me? I'm desesperated.

Thanks

do your product/colony digestions appear to be supercoiled? perhaps the sites are destroyed and you are not getting linearized construct after you digest? Before repeating the whole thing, I would digest with an enzyme (not XhoI) that will linearize the constructs...only one cut...and see what you get, in case you have the right thing

also, could you perhaps post a pic of your gel?

I don't understand what are in the different lanes of your gel? could you please label with more detail?

all I can say from your gel is that it looks like your digestion is pretty complete, as near as I can tell

I've had luck with Xho I sometimes, but sometimes not...don't know why, but it seems to occasionally be troublesome when used for cloning. I theorize that there are/is an unknown property of the enzyme that can affect downstream applications...

anyways, please let me know what's in each lane

and also, to clarify something you wrote in your ?, is your first insert 4kb or 5? as it's written, it appears that you're getting a 5kb insert from a 4kb plasmid? I'm certain that I may be misinterpreting your words...

good luck

So, for the gel
Lanes 2,3,11-15: it's Xho1 digestion for miniprep of the 5 kb fragment and bgal vector ligation.
Lanes 4-10: it's Xho1 digestion for miniprep of the 3 kb fragment and bgal vector ligation.

Also, my first insert is 5 kb and the size of the vector pCRII is 4kb. So the total size of the plasmid is 9kb.


thank you so much, again, to help me with the mysterious cloning.

what about the sizes of your standard bands?

I also think you are not getting complete digestion

can you post a pic of when you cut with different enzymes to linearize? and, did you include a vector digestion control for comparison?