choosing competent cells - (Aug/16/2007 )

Hi
I am trying to clone an 750pb insert in a 20kb vector for one month and an half, but with really no success.
The strategy is to isolate the GFP (750 pb) from the plasmid PCMS-GFP by PCR, then to clone it in a 6kb vector containing a 15kb insert. Both primers where designed to include the Nhe1 restriction site (present one time in the vector), adding this site on both extremities of my insert. The insert has just one nucleotide after the restriction site, which could have been a problem to cut the DNA. But NEB says that the efficiency of cut for Nhe1 with only 1 nucleotide before the site is 100%. Furthermore, I had successful ligations (I mean I successfully inserted the entire insert in part of the vector, the size of the resulting plasmid is another story), and observed dimmer and trimmer of the insert when I ran the cut insert on a gel.
The vector is cut with Nhe1 during 6 hours, to assure complete digestion. I indeed don’t separate my plasmid on a gel to minimize the exposure of DNA to UV and agarose. The vector is then treated with CIAP to avoid any religation.
I tried to do the ligation over night at 16C, or other the week end at 4C. I tried several insert/vector ratio (2/1, 3/1, 4/1, 5/1, 10/1)
I’m using one shop top ten cells from Invitrogen, which are the DH10B strain, for transformation.
So far I checked the following :
-My PCR product is of the expected size, I don’t amplify anything if I don’t put a template in my reaction, or if I put the vector as a template.
-Nhe1 cut the vector only once, even after 6 hours of incubation
-if I do a ligation/transformation with only my vector, I don’t have any colonies.

I got colonies with the ligation product, but when I cut the plasmid obtained with Nhe1 and run it on a gel, I see one band at 750 pb (corresponding to the GFP) and one at 6000pb (which would correspond to the vector’s backbone without the insert).
If I do a PCR on the resulting plasmid using the primers used to isolate the GFP from PCMS-GFP, I see one band at 750 pb (the expected size).
I do think that I may have a recombination of my plasmid inside the bacteria. Should I change for a strain specialized in big plasmid? Or in unstable DNA?
Thanks

Hi,

have you had any success with your cloning? Which bacteria strain did you take?

I think I have the same problem as you in my cloning.

Thanx

[Sorry for the delay.

No, I still got no success with my cloning. I keep trying...

did you gel purify your insert? May I ask what is the size of the PCMS-GFP plasmid?

Also, what is the vector backbone that you are using? Is a low copy plasmid or a BAC of some sort? And what is the nature of your insert? Is it a repeat array?

Hi again,

I used XL1-Blue Supercompetent cells (chem comp) from Stratagene for adenovirus plasmid (~34kb) transformation and it worked. But I am sure there are other strains special for big plasmids.

Cheers

Hi.


[quote name='perneseblue' date='Sep 7 2007, 03:00 PM' post='111068']
did you gel purify your insert?

I was not doing it, since I read that it may inhibit the ligation. But after somebody told me to do so, I tried again. It seems to work that time, I have the good size of bands.

May I ask what is the size of the PCMS-GFP plasmid?
5.5kb

Also, what is the vector backbone that you are using?
PCDNA3.1 (-)

Is a low copy plasmid or a BAC of some sort?
It is a low copy plasmid

And what is the nature of your insert? Is it a repeat array?
No it is not.

I seem to have got my ligation this time. Basically I gel purified my PCR insert, and I grew the bacteria longer at 26 C. I got some fast growing colonies that were not containing the expected plasmid, and some slower growing colonies that seem to contain the expected ligation product. I don't know if the problem came from the fact that I did not gel purify the plasmid (I would think so), or from the fact that my bacterias were growing too fast and expelling the plasmid, or only the one with smaller plasmid were growing... Sorry it may sound stupid, but I'm just starting in molecular biology.

I'm testing the plasmid this week.


Thanks