Plasmid stability/Cloning problem - (Nov/07/2006 )

Hello

Can someone provide us any pointers to this problem:

We are cloning a 1.3 kb fragment into a 6.2kb vector. The vector is pNEBR-X1 and the insert is a mammalian protein generated using PCR. We digest the vector with XhoI and NheI and phophtase treat it prior to ligation. Our blank ligation plates give us ~10 - 15 colonies while our ligation (3:1, 1:1) give more than 300 colonies. However, not a single colony seems to have the right plasmid. We have screened approximately 20 colonies from each ligation (~ 150 total). We do mini-preps and when we digest with XhoI and NheI, we always get a dark band which is ~ 3 kB. More importantly, we do not get a 6.2 kB band corresponding to the vector. Digesting with RE other than the ones we used for cloning also gives similar results. We have also tried the following: different batch of ligase, different prep of competent cells, different tube of enzyme - does not work. We have checked our vector and insert before ligating and we get a single band.

I though it could be a plasmid stability issue but NEB says they have no data on stability.
1. Will the host strain impact stability? We are using E. coli TG1.
2. If it is a stability problem and we are losing part of the plasmid, is it possible that the SAME portion of the plasmid is getting lost each time? We always get the ~ 3 kb band..

Greatly appreciate any pointers

Arul

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Could you elaborate. What were the restriction enzymes used. The expected product and the actual product.

Could it at all be possible that you are looking a double inserts? 2x1.3kb is quite similar to 3kb......(The none horror answer)




However I am worried about the use of TG1 as a cloning cell. TG1 is recA+, end+, gyr+....
You get the picture, every endonuclease and recombinase normally knocked out in a cloning strain is active in this strain. So yes, plasmid stability is severely affected.

2. Yes. There might be a sequence which looks somehow familiar (sequence wise) to the cell, so it will recombine (even cut since your endonuclease are active!) within that region.

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Hi
Thanks for the message.
We use XhoI and NheI as the RE. The expected product is 1.3 kb. The product we are getting is ~ 3 kb. Moreover, we do not see a band corresponding to the 6.2 kb vector.
I think you are right pointing out the cloning strain as a likely issue. We will try HB101 (recA-). Do you have any suggestions on the strain?
Thanks a lot

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Suggestion on HB101...

HB101 genotype
F- mcrB mrr hsdS20(rB- mB-) recA13 leuB6 ara-14 proA2 lacY1 galK2 xyl-5 mtl-1 rpsL20(SmR) glnV44 λ-

I have never used this strain before. But looking at the genotype listed in OpenWetWare, HB101 is certainly far better then TG1 expression strain. recA and hsd have been knocked out..... but endA still active... so you might have to be quick on the miniprep to avoid degredation on the plasmid DNA.

Well it is okay and will probably do... just not the best cloning strain around.

As for personal perference I like XL1-blue. It maintains plasmids with large tandem arrays relatively well. And best of all it is one several of E coli strains that has low liposaccaride synthesis, so it is great for colony PCR. Down side it is a bit slow to grow.

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