8 replies to this topic

[molbiouser]

I have been trying to do a simple subcloning reaction. I am moving my insert (1.2 kb) to a FLAG tagged vector. Both are cut with EcorI/XhoI. The vector is also dephosporylated with CIP or Antartic phosphatase (NEB). Ligation reactions are done with quick ligase (NEB or Roche) or the T4 DNA ligase. In all cases I get a lawn on bacteria from transformations from empty vector, and the insert+ vector. Could someone help me out here.

[rassen]

Have you purified your vector after digestion to get rid of the smalll fragment and prevent the vector from ligating back?

[molbiouser]

the vector was gel-purified after digestion with Ecor1/Xho1, and then column purified after digestion with CIP.

[CKM]

Are you sure that both of your enzymes cut the vector (check correct buffer composition, digestion time)?
If in doubt, you could digest with your first enzyme, clean up and/or adjust the buffer and digest with your second enzyme.

How do you do the transformation? Electroporation? Do you re-use the cuvettes?

Do your agar plates contain the appropriate antibiotics? Perhaps they are too old?

Greetings
CKM

[wirly]

Usually the only way you get a lawn from a ligation (particularly the control) is from not having any/enough antibiotic on your plate or using a bacteria with that antibiotic resistance.  

You may also get very high density with electroporation ausing an uncut vector if you plate a lot of the transformation.

Make new plates, make sure your bugs are OK and do it again.

[molbiouser]

I generally use the chemically competent cells DH5alpha (Invitrogen). I had changed my plates (LA-AMP) very recently however the pattern is the same, a lawn with empty vector as well as ligation reactions. Normally from ligation reactions I plate out the entire transformation mixture.

For cutting with EcoRI/XhoI i also cut another bector having my insert with the same enzymes and it worked fine so I assume that the empty pFLAG vector too would be cutting fine with the two enzymes.

Thanks for your help!

[wirly]

molbiouser, on Aug 12 2004, 12:13 PM, said:

For cutting with EcoRI/XhoI i also cut another bector having my insert with the same enzymes and it worked fine so I assume that the empty pFLAG vector too would be cutting fine with the two enzymes.

That seems logical, but if you can get vector back after growing up and minipreping some "lawn" then you can't be getting digestion of the vector for some reason, especially since you also CIP it.  

It's also not endogenous resistance in the bugs since you can get vector back.  



Now for the lame questions...How much vector did you cut with how much enzyme in what volume?  Did you see a nice single vector band in the gel purification?  How much vector did you use in the ligation?  Did you accidentally take vector from the uncut vector tube instead of the cut.  This has been the cause of much head scratching on an occasion or two with people in various labs I've been at.    

[InvisibleSurfer]

I think what causes this is:

1. When you cut the band out of the gel, you are also cutting undigested vector (there's ALWAYS some undigested material). the solution to this is to run the undigested, original plasmid next to the digested vector and make sure you are cutting at an entirely different site (if possible). Also, run a high percentage gel, >2%

2. Antibiotic; I've fucked up this bit big time and took me a year (!) to realise. For ampicillin (or carbenicillin), make a stock of 100mg/ml (and use at 100um/ml) by weighting out 1 gram and resuspending it in 10 ml. The mistake I was making was that I was calculating the MW of amp, ending up with 100mM instead of 100mg/ml...

[wirly]

Yes, it's true there is always some undigested material, and often it is so close in size that it is excised along with your desired band, or your material drags some uncut along with it.  There is no way that little uncut is causing a lawn no matter HOW competent your bugs are unless you are doing something wacky like growing them o/n in the recovery stage before you plate.

Also, higher percent gels are usually done to distinguish smaller particles, lower percent to separate vector sized ones...at least in my experience.