Cloning into expression vector - Why doesn't it work anymore? (Aug/01/2006 )
Hi there,
I am trying to clone a gene into an expression vector (CMV4).
But unfortunately it doesn't work anymore (I did the same for another gene a half year ago and everything was fine). I don't know what could be the reason. Maybe someone of you has any suggestions?
Here is what I've done:
First I cloned my gene into pTOPO-vector and checked the sequence. Then I cut the insert out of the vector (using HindIII and BglII - I had used Primers with those restriction-sites to amplify my insert) and purified it by gelextraction.
The CMV4-vector I cut with HindIII and BglII, too, and dephosphorylated it (Antarctic Phosphatase, NEB). Then I purified it with the NucleoSpin-Kit (MN).
Then I tried to ligate (T4-DNA Ligase, NEB) and added some NotI (and BSA + RE-Buffer, of course), too, because this one cuts between my used restrction sites and should hinder religation of the vector.
Finally I transformated into TOP10F'.
But nothing grows. 
I checked the Ligation by using another Ligase (QuickLigation, NEB) -> Nothing grows.
I chekcked the cells using another sample -> they are okay.
I didn't put the NotI into the Ligation -> colonies grow but have no insert.
Well, I don't know what to do now. What is going wrong? 
Seems like my insert doesn't want to go in there...
But after all it already worked a half year ago...
I really hope you have some ideas for me.
Thank you!
Chakchel
everything seems just fine. but u cannot be sure that your vector is absolutely cut by both of the enzymes, can you?
i have had that problem many times. did you perform a double digestion or did u cut your vector with the enzymes one by one.
i have had that problem many times. did you perform a double digestion or did u cut your vector with the enzymes one by one.
Thank you for that fast answering!
I did it already both ways... well, even three ways: Double digestion; first HindIII then BglII; first BglII then HindIII
A sample before the second digestion step showed that the enzymes worked properly.
But still there was nothing growing when I used these vectors for ligation
Chakchel
how many bases are there between two restriction sites in MSC of vector? both of your enzymes may be working, they may both linearize your vector but they may not cut the linearized vector. at least this was the case for me, did you add double digested vector self ligation control to your ligation reactions?
There are 22bp between the restriction sites. Might be enough, I thought... but maybe it isn't.
Religation I saw when I used the Ligation without NotI (see above...). I didn't check the sequence, but I guess you are right and they are only cut once.
But the strange thing is that I used the same vector and the same enzymes a half year ago - and got plenty of positive colonies...
Hmmm, I have a new idea now!
What about cutting my old insert I cloned a half year ago out of the plasmid and then ligate again using the "old" vector?
I should see if there is successful double digestion on gel then.
I will have a try... Thank you very much for giving me your thought-provoking impulse!
I will write soon whether it worked that way...
good luck
Hi!
I am really happy to write that it now worked.
Lots of positive colonies!
Greetings,
Chakchel
hobbareyyy
congratulations!