Plasmid lost during culture - (Jun/17/2008 )

I might give that a go Zek, or at least analyse the eluants in each step of the miniprep procedure, just to make sure i'm not losing it somewhere. Hard to believe that that DNA would be lost when others are fine though.

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Hello,
I have a similar problem, maybe more weird. Sorry, this might be too long, but please listen
I want to generate mutated ryanodine receptor construct (21 kb). I have to full-length rabbit RyR1/pCI-neo plasmid, OK.
For site-directed mutagenesis, subcloning framgents (~3 kb) to an adapter-equipped pBlueScript was no problem, sequencing and restriction map confirmed the success.
Ligating back to the full-length, this's been making me crazy now for months
I cut with the right enzymes the full-length and the subclone. Use BAP. Isolate with gel-running, purify with Promega kit. Confirming on gel: expected length, plenty of DNA. Ligatin... a "generic" Takara ver 2.1 did not really work, I switched to the Takara Ligation kit LONG, that seems to be nice. I got many colonies, comparable to the positive control, and only 2-3 from negative controls: ligating only vector and only insert. Colony PCR showed promising signs: I could amplify fragments from various sites of the 21 kb plasmid and also from the two ligation sites (ie: around base 1500, 7600, 14000 and so on...)
The real problem stars now:
If I culture colonies for screening, I got DNA only in ~10% of the cultures. There are E coli (XL10 Gold), but no DNA. If there is DNA, after purification (Promega SV Wizard Miniprep) the size of the reulting plasmids are about only 7-8 kb!! And from these I cannot get the above-mentioned nice confirming PCR pattern. Naturally, the 7 kb long something cannot span the ampicillin resistance gene together with the 14 kb long confirmed sequences.
If I spread colonies to a new plate to get large ones to collect somehow, the PCR result is the same, but so far I could not get DNA from these ones. Difficult to suspend the cells... So I think something happens to the cells in the liquid culture. But what?
I have been struggling with this for 4 months and I have 4 weeks to get something. Any bright idea? I would be extremely thankful!

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Sorry, I am not going to go into detail about other things, but one thing to keep in mind is that if you have a plasmid >10Kb size, transformation by heat-shock is a very inefficient method. You need to electroporate in such case to get a good number of correct colonies.

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Thank you. However, I forgot to mention, that I have no absolutely no problem with the original (not made by me) full-length RyR1... always get plenty colonies by heat-shock, and no plasmid loss or fragmentation during liquid culture. And I think that the transformation is quite good, for the aforementioned promising PCR-pattern can be gotten from virtually all colonies.
But I give it a try.

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It is different matters altogether to transform an intact plasmid (high conc, supercoiled, clean), and a cloning reaction (very little conc of intact circularized correctly ligated plasmids, not-supercoiled, dirty). So, your orignial plasmid may give many colonies by heat-shock despite low efficiency of transformation, but not your ligation reaction. You need electroporation.

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Oh, maybe right. But as I said, until the colonies, everything is OK. I have plenty colonies, most of them seems to have the plasmid, confirmed by colony PCR. If I replate (or what) them to a new plate (as in the case of the master plate for col.PCR) they grow happily.

Or even if they have the plasmid, it is not sure that it is capable of proliferate efficiently in the bacteria?
Moreover, as I said, I have cultures for screening but I cannot get DNA from those, as if it had been lost during the culture. Or maybe it is a contamination but I have negative controls for that.
I ordered SURE cells, maybe those... until then, any new idea?

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I'm looking into this now ryanodine. Can you give me the sequence of your complete and mutated gene? I suspect it is a sequence thing. There is another poster with the same problem at the moment. I'm going to compare all our sequences.

http://www.protocol-online.org/forums/inde...mp;#entry141075

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Wow. OK, I send you two (attached), but the problem is that I have six different mutated plasmid... with the same disappointing result, and the parent, original RyR is OK. Of course, there is the possibility that something wrong with the ligation itself, but I think that the colony PCR is against this.
Thank you very much for your effort.
Maybe the cause of this wider-spread problem is related to the disappearance of the bees...

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Can you send me just the sequence of your complete gene and the mutated genes - not the vector. The vector is the same but the genes are different so that's what i'm interested in.

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Dear killerkoz,
Sorry for not answering, I was on holiday.
I attach the sequence to this post. RyR1 is the 21 kb gene. I, however, do not give you a separate file with the mutated ones, because I introduce 6+ different mutations (for localization, please, see ryr1_mut file), to different locations, and the result is the same. no correct colony or no growth.

The other issue: ryr1_c-term: subclone is the c-terminus (~4 kb) of ryr1 in pEGFP-C1 vector.
I want to introduce mutations with site-directed mutagenesis (PCR method). I invariably get a 200 bp shorter fragment between 1300 and 3500 if I cut with NcoI, independently of the site of the mutagenesis, i.e. within or outide of this region. I know that it is PCR, but it is at least strange that the problem is always the same. I tried longer and longer extension times, but from 50 tries I got only once a correct mutated vector
Can you tell me if there is any difficult sequence there?
Thank you in advance!

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