Plasmid-Stability? Fragment-Deletion? I'm in dire need to identify and solve - (Oct/19/2010 )

TGS on Wed Oct 20 14:07:03 2010 said:

For site directed mutagenesis you can excise the fragment where the mutation should be(make it as small as you can), put it in a small, high copy cloning vector (like pBluskribe) and do your rolling circle pcr on that. Select the correct clone, expand, isolate your fragment and put it back in your vector. Easy:)

what i just saw from his sequences he sent me is that he has a direct repeat with 94 bp that could lead to a possible deletion of 1094bp ...this can happen as well by recA-independent recombination (but with a less lower frequency i assume).

Using more stable strains like Stbl could maybe help!

Best regards,
p

Seems like the 30°C Transformation didn't work. I incubated it with LB for around 1h15min at 30°C before putting it on the plates and letting it grow at 30°C and can't see anything remotely like a colony on the plates. Will I still have to wait or did I incubate the cells not long enough?
How do you guys perform a 30°C transformation?
(As control I did a normal one with the same amount of DNA at 37°C which yielded quite a lot colonies)

If your antibiotic is amp, i don't see any easy explanation for the lack of transformants. If you have a different selection antibiotic in your plates, then outgrowth at 30C may not have been long enough for resistance to be expressed in your cells

Have you considered that the problem might be that the construct you are attempting to build is very toxic to the cells? You may be carefully selecting for versions of your construct which are wrong and (therefore) non-toxic.

A 1094 bp deletion would go out of frame, so it might remove the toxicity of a protein coding region.

z-hunt values are as well very high with his inserts ...maybe its the cause for the instabilities!

z-DNA is known to be unstable in E. coli. For those who are interested see this link!

Best regards,
p

I just wasn't patient enough. The 30°C plates start showing small colonies... thought I'd see the first traces of them in the morning, but apparantly they need much more time to grow in comparison to 37°C than I first expected... If I'm lucky I can even pick some before leave in a few hours...

Neither the 37°C nor the 30°C incubation yielded any of the mutated plasmid I am looking for (I tried 12 colonies of each one of them). Next thing I'll try is picking another 24 colonies of the 30°C plate and incubate them for 2 days at 30°C before plasmid preparation. Don't think this will make a major difference, but in the 12 colonies there were two which weren't as small (but still too small) as the other ones... maybe this is a sign that here the tendency for bigger plasmids is actually higher.

pDNA on Thu Oct 21 14:09:26 2010 said:

Nice find! From the paper I understand in smaller plasmids a part of Z-DNA forming inserts is recombined (even in RECA1 genotype- DH1) after incubation in bioreactor, but unaltered plasmids remain. I wonder what happens for plasmids of larger size. Perhaps there is just a small part left that is unchanged. Still, it's strange that he can't even find a few clones with the expected plasmid.

A quick look around suggests stratagene offers a strain with increased z-dna stability (SURE, SURE2) (http://openwetware.org/wiki/E._coli_genotypes, http://cp.literature.agilent.com/litweb/pdf/5989-8295EN.pdf).

To me it seems also strange that all clones are negative ...if it is due to recombination it must be a really nasty sequence feature!

The only thing to elucidate if it is really due to recombination would be sequencing of the plasmid and look for the exact deletion events and check if they are flanked by some repeats or Z-DNA stretches ...but that's laborious and does not really help you in the course of your project since you are looking for functional plasmids

Another thing that comes to my mind is:
Maybe what you get is a combination of some recombination and low efficiency in ligation? ...you are cloning long fragments of DNA (10kb) ...what ligase ara you using? Normal T4 Ligase is not very efficient in cloning large fragments.

Maybe you can use a ligase that is known for good efficiency with large fragments like this one.

Maybe this one does the trick

Best regards,
p

hematopoietry on Tue Oct 26 09:58:32 2010 said:

I'd guess as soon as a smaller plasmid with the resistence is formed it will have a major advantage since it means less metabolic stress to reproduce and also allows faster reproduction. This way it becomes the dominant form in a colony.
One of my latter expression showed something what could be a trace of a higher band (even in digestion) at the expected size. I recently made a gel with lots amount of plasmid of two of these samples and cut them out. I'm thinking about retransforming them and prepping them just to see if these were different Iso-forms or if in this case some of the original plasmid remained... but I'm rather pessimistic.

What bothers me most is that here in my lab this plasmid was already several times succesfully mutated (even though other mutations were done), so it seems it's not a general problem of this plasmid, although one can see that in the older mutation experiments also some plasmids appeared which shared characteristics with my problem (a short fragment which fits fine and another fragment which is way smaller than it should be).
I really tried a lot of things yet like skipping dialysis, trying other cells (XL1 Blue instead of TOP10) incubating with lower amounts of antibiotics, incubating at 30°C and also picking a plate completely empty... all which yielded no results... the next and probably last thing I'll try (at least my other projects start to running more smoothly) is using NEB10beta cells and using heat shock for transformation. Since I always good good amounts of colonies I'm optimistic that the lower transformation-efficiency won't really bother me...


P.S.:
Last week I created two plasmids (2.5 and 3 kb in pET41a -> 7.5 and 8 kB) on the first try... so it seems two be really a specific problem of these constructs and rather not of myself or the materials I use.