Need some help! - (May/22/2006 )
I'm trying to clone a 3.8kb fragment into pcDNA3.0 (5.4kb).
I've used pGEM-T vector (3kb) from Promega and have successfully cloned my insert into the T vector.
I then amplified the plasmid, pGEM-T-insert, using a Midi prep from Qiagen and double digested 20ug of the plasmid with EcoR I & Xba I. Meanwhile, I've digested my destination vector with same enzymes. After gel electrophoresis (95V, 1hr50min), I've cut out the insert & vector, followed by gel purification. I dephosphorylated my vector with CIP (10U/ul, 0.1ul) from NEB for 30min and then purified with PCR purification kit from Qiagen.
I did ligation as such: 7ul insert, 1ul CIPed vector, 1ul Buffer & 1ul T4 DNA Ligase. (16hr, 16 degree) I did a control reaction as well: 7ul ddH2O, 1ul CIPed vector, 1ul Buffer & 1ul T4 DNA Ligase.
I then transformed 5ul of my ligates (both standard & control reaction) by heat shock.
The results of my plates seemed to be wonderful: no colony for my control, whereas ard 100 colonies for my standard reaction.
However, after I have selected 12 colonies, extracted plasmids and double digested with the same two enzymes, I've found all of the colonies are actually formed of pGEM-T-insert.
I think the problem came from gel purification step. However, I've tried to cut the desired band as slim as possible.
Hope somebody could help me. Thanks a lot!
Yes, most certainly it is a problem of seperating pGEM-T from your insert on the gel (3.0 and 3.8 kb). I thought once that I had cut the insert (2,7 kb) out properly, but still had a contamination with pGEM-T in my new colonies.
One thing that helps is cutting your plasmid first with an enzyme that only cuts the vector, then cut the plasmid with EcoRI and XbaI. So you will find your band at 3.8 kb and pGEM-T fragments at lower sizes.
What I don`t understand is why you don`t see any colonies with pcDNA3.0 + insert.
I ended up with a funny mix of insert, pGEM-T and pcDNA3.0. I mean all possibly combinations ... ;-)
At what size runs the uncut plasmid (pGEM-T-insert)? At ~ 4kb? ...
I would also make sure, that you don´t have a contamination of pGEM-T-insert in the ligase or in the ligase buffer.
Good luck!
U have started with quite a bit of DNA 20ug, so you would have to digest it for sufficient amount of time so that all the molecules are digested.
After you digest it again, run it on the gel longer so that you will have better seperation of the uncut and cut DNA. Now you should be able to cut the insert without much problem.
If you think the 2 bands are closer, run it longer, get a decent separation (as long as you can cut without disturbing the uncut) and go ahead.
Good Luck !!!
in addition to running it longer, i would also regulate the gel percentage.
-viv
One thing that helps is cutting your plasmid first with an enzyme that only cuts the vector, then cut the plasmid with EcoRI and XbaI. So you will find your band at 3.8 kb and pGEM-T fragments at lower sizes.
What I don`t understand is why you don`t see any colonies with pcDNA3.0 + insert.
I ended up with a funny mix of insert, pGEM-T and pcDNA3.0. I mean all possibly combinations ... ;-)
At what size runs the uncut plasmid (pGEM-T-insert)? At ~ 4kb? ...
I would also make sure, that you don´t have a contamination of pGEM-T-insert in the ligase or in the ligase buffer.
Good luck!
chalet2, thnx a lot! I'll try ur method. And I'll use a new tube of ligase buffer.
I think i didn't make it clear. e plate for control reaction (only dephosphorylated vector inside) got no colony, whereas e one for standard reaction got ard 100 colonies (almost all are formed of pGEM-T-insert).
I'll post my results once come out.
After you digest it again, run it on the gel longer so that you will have better seperation of the uncut and cut DNA. Now you should be able to cut the insert without much problem.
If you think the 2 bands are closer, run it longer, get a decent separation (as long as you can cut without disturbing the uncut) and go ahead.
Good Luck !!!
Thnx to scolix. I think i'll digest overnite instead of 2hrs next time.
I'm a bit confused abt e uncut & cut thing. e plasmids are completely cut visually. (there might b trace amnt of uncut plasmid, but I dun think it can b visualized.) so how can I make sure uncut & cut bands are separated?
-viv
I'm using 0.8% gel. wat percentage would u suggest?
You might not be able to differentiate trace amounts of uncut DNA from cut DNA.
The idea is to have a good separation on the gel to really separate the 3kb and 4kb marker bands and then should be able to c ur 3.8kb insert close to 4kb and now you cut the insert out, which hopefully does not contain any uncut.
I usually differentiate between cut and uncut DNA by looking at the shape of the DNA bands. If the band has a smooth curve on the side its running (positive side of the running chamber), then it should be cut.
Good luck !!!