empty vectors more than the insert - (Aug/06/2010 )

hi

I have cloned a human transmembrane protein into a vector and did the transformation. On the plates there are lot of colonies, but when I screen the colonies, Isee only the empty vector and not with the insert, though the gel of the ligation shows the required band. Is there any way to increase the efficiency so that I get the insert more than the empty vectors. I use a ration of 1 and 3 for the vector and the insert. I have done many transformations, but everytime the same gets repeated again. not even 1 in 50 are positive. Please help.

Jaya

Which enzymes are you using to cut your vector? Did you dephosphorylate the vector?

Stardust

jaya2020 on Fri Aug 6 12:20:06 2010 said:

No I did not dephosphorylate it. No one in the lab uses it. So I did it without dephosphorylation. I used Xba I and Cla I for the digestion. It was a PCR product with these restriction sites. Help me if you can. So desperate for help.

jaya

You need to tell us much more about exactly what you did.

Did your PCR primers have 5' overhangs past the restriction site?
Did you purify the PCR product before cutting with the enzymes?
Are you sure the XbaI vector site can be cut (no methylation issues?)

We can't help unless we know exactly what you have done.

First you should try to find out where the empty vector background is coming from (ie: incomplete digestion or re-ligation). Transform the prep of vector you are using in the ligation reaction without ligation. If this gives you colonies then your digest was not efficient and you need to redigest/purify. If you get little or no colonies with this transformation, this means your background is through re-ligation. You should check to make sure each enzyme is digesting properly by doing single digest of a small amount of your vector. You should also dephosphorylate your vector. Many people don't do this and it isn't absolutely necessary, unless you are having empty vector background problems. Next, have you run a small amount of your vector prep and insert on a gel to visualize them? You should run a small amount with a quantifying ladder just to make sure the concentrations you think you have is indeed what see in a gel. In my experience, this is a large source of problems. People do an OD260 and think they have lots of insert but when they run it on a gel, turns out there is only a fraction of what they thought. Finally, set up a ligation with a higher insert ratio (you are doing molar ratio, right??). I've had to go all the way to 10:1 insert:vector to get a ligation before. How are you ligating? What enzyme, how long?? I always recommend the T4 (not quick ligase) for four hours at room temp in a 20 uL reaction, transform 2-4 uL and then let the rest sit overnight on your bench. If the plates don't show you a difference compared to the no insert control ligation then retransform. Also, if the plates don't have colonies (after you troubleshoot and fix your empty vector background), throw them back in the incubator, especially if your selection in Kan. You've got nothing to loose and I had a labmate only (finally) get his clone after he forgot his plates and let them incubate for two days. Turns out for whatever reason, the bacteria grew very slowly with his construct.

rkay447 on Fri Aug 6 15:14:09 2010 said:

Not only XbaI can result in problems due to methylation but also ClaI. Are you using a dam/dcm negative E. coli strain to get your vector backbone?

XbaI can not cut if you used normal E. coli instead of dam/dcm negative strains if GA is in front or TC behind the restriction site.

ClaI can not cut if you used normal E. coli instead of dam/dcm negative strains if G is in front or C behind the restriction site.

In either case you could get vector that is only cut with one enzyme. In this case you will get a lot of religated plasmid. Best choice would be a dam/dcm negative strain like like ER2925 NEB.

Moreover, the enzymes can only cut your PCR product effectively if you add some addotional bases, e.g. I always add 6 bases: 5' cgtcag-restriction site-primer sequence 3'

Sometimes PCR product digest is not very effective even with overhangs. In this case I subclone the PCR product either in pGEM Teasy (Promega) or pJet2.1/blunt (fermentas) and then digest with the enzymes. This way you have more control over it because you can visualise it with a gel and cut the insert band out to purify it.

In worst case I set up a ligation like this:

2 µl 10x ligase buffer
1 µl T4 DNA Ligase
100 ng vector
Fill up with insert to 20 µl

Incubate RT for 2 h, the O/N in the fridge, the electroporate 2 µl into 50 µl competent E. coli.

Stardust