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Problems with STBL3 cells - Digesting DNA from STBL3 cells (Nov/29/2005 )

Hello,
I am cloning into a lentiviral vector. I transform my ligation into STBL3 competent cells, pick and grow colonies, do minipreps, and check for my insert with restriction enzymes. When I run the undigested miniprep on a gel, I see DNA. When I run the digested DNA, I see nothing. Transforming the same ligation reaction into DH5 alpha cells and using the same restriction enzymes, this does not happen.
Has anybody experienced this before/found a solution?
Thanks a lot!

-aaw-

[quote name='aaw' date='Nov 29 2005, 06:36 PM' post='32909']
Hello,
I am cloning into a lentiviral vector. I transform my ligation into STBL3 competent cells, pick and grow colonies, do minipreps, and check for my insert with restriction enzymes. When I run the undigested miniprep on a gel, I see DNA. When I run the digested DNA, I see nothing. Transforming the same ligation reaction into DH5 alpha cells and using the same restriction enzymes, this does not happen.
Has anybody experienced this before/found a solution?
Thanks a lot!




I also have this problem when I am making this clone, it is the exactly same thing as you had. What should I do? I feel very depressed.sad.gif sad.gif

Do you think Transfect the LR reaction sample into DH5a is Ok for the lentivirus clone? Do you find the method to solve this problem?

Thanks.

-BLUE-SNOW-

If you're using STBL cells, you're doing this because you're having unstable sequences (like lentiviral long terminal repeats), right?

I've used STBL2 cells with good results, but most of the time I'm just using DH5a. If you're afraid of losing your LTR's or so: grow them @ 30°C and shaking @ 180 rpm. Also: when transforming use these conditions and incubate LB plates @ 30°C. Has worked very fine for me. Takes a couple more hours to grow your bacteria, you get smaller colonies (but you also decrease your chance of sattelite colonies if you tranform and cannot be back next morning). The reasoning behind this is that somehow you would 'slow down' or decrease e. coli metabolism.
Don't know if it's true, but I've used these conditions to grow large (more than 15 kb) plasmids containg entire HIV genomes, including the entire 2 LTR-regions.

-vairus-

I placed this answer already in another topic. So repeat. According to Invitrogen:

"This strain is endA+. A very thermostable periplasmic endonuclease will co-purify with the plasmid
DNA, possibly shearing the plasmid during restriction enzyme digestion since the endonuclease needs Mg for activity. The procedure should be terminated with a phenol/chloroform extraction or purifying the plasmid DNA on a column. If making solutions I, II, and III (alkaline Lysis, see Maniatis or Current
Protocols for Molecular Biology) for a conventional DNA prep procedure, make sure EDTA is present in
solution I."

"Different-sized colonies and some mini-preps that show LTR recombination may be observed when using the Stbl3 cells. If this occurs pick several colonies of different sizes (i.e. at least 5 small colonies, 5 medium-size colonies, and a few large ones) and analyze them with restriction enzyme digestion. Another option is to do double antibiotic selection on the LB plates with both Amp and Bsd. Remember: first recommendation is selection with 100 ug/ml Ampicillin"


So, try this

-Taras Bulba-

For lentiviral vectors I use Stbl2 for long time storing and otherwise transformation and stuffs work fine with XL1Blue.(and yes I grow stbl2 cells at 30C)

-Calvin*-

I have the same problem.

-lonemen-

We have the same problem, but we usually use higher ampicillin conc. to overcome this problem and always use the columns.

-scolix-

Hi!

I've used STBL2 cells for the transformation of LTR-like constructs, but also for cosmids. By accident, I ordered STBL3 cells instead of STBL2. Does anyone know if there is a difference in capacity of those 2 different competent cells? I cannot find the info on the invitrogen website...

Thanks!

-brigitta-