Unstable clones

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pjansen
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Unstable clones

Post by pjansen » Wed Apr 19, 2006 9:03 am

Hello, I'm trying to make a plasmid containing a huge hairpin (500bp). When I'm testing my plasmids about 15% of them has deleted te hairpin-structure. Does anyone have any suggestions, like competent cells, etc.?
Thank you very much.

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LilKim
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Post by LilKim » Wed Apr 19, 2006 5:26 pm

Maybe i'm not undertanding your question... but, i think that problem should be eliminated when you're doing your mini-prep.

Many "wacky" things can happen whe you're transforming plasmids... that's why you should always check several colonies.

When i do it .. i'll:
1. Plate e.coli on selective media.
2. grow up single colonies.
3. (maybe) set up and overnight culture. ( pick 8-12 clones, sample!)
4. Mini-prep.
5. You can then run out 1-2 microliter of pDNA from each clone to make sure you have the right size plasmid (and to eliminate the clones that have deleted the hairpin).
6. Send several of the remaining off for sequencing (to determine directionality of the insert).

Cloning has not been the most efficent thing for me...

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LilKim
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Post by LilKim » Wed Apr 19, 2006 9:10 pm

Maybe i'm not undertanding your question... but, i think that problem should be eliminated when you're doing your mini-prep.

Many "wacky" things can happen whe you're transforming plasmids... that's why you should always check several colonies.

When i do it .. i'll:
1. Plate e.coli on selective media.
2. grow up single colonies.
3. (maybe) set up and overnight culture. ( pick 8-12 clones, sample!)
4. Mini-prep.
5. You can then run out 1-2 microliter of pDNA from each clone to make sure you have the right size plasmid (and to eliminate the clones that have deleted the hairpin).
6. Send several of the remaining off for sequencing (to determine directionality of the insert).

Cloning has not been the most efficent thing for me...

pjansen
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Post by pjansen » Thu Apr 20, 2006 8:07 am

LilKim wrote:Maybe i'm not undertanding your question... but, i think that problem should be eliminated when you're doing your mini-prep.

Many "wacky" things can happen whe you're transforming plasmids... that's why you should always check several colonies.

When i do it .. i'll:
1. Plate e.coli on selective media.
2. grow up single colonies.
3. (maybe) set up and overnight culture. ( pick 8-12 clones, sample!)
4. Mini-prep.
5. You can then run out 1-2 microliter of pDNA from each clone to make sure you have the right size plasmid (and to eliminate the clones that have deleted the hairpin).
6. Send several of the remaining off for sequencing (to determine directionality of the insert).

Cloning has not been the most efficent thing for me...


Thank you for your reply, the problem is that when I select different single colonies and grow them seperately, each single clone appears to delete some hairpins. Even when I have a 'positive clone' and I start up a new growth from the glycerolstock, this positive clone will delete the hairpin in some bacteria (or maybe worse, within bacteria), so the result is a mixture of plasmids with and without the hairpin. I can easely check this with restriction analysis.
Maybe some more details: I've used Statagenes SURE2 strain, which is a special strain for 'unclonable' DNA and I also tried to grow my cultures at 30 C in stead of 37 C.
I hope someone can get me some advice for this.
Thanx

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LilKim
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Post by LilKim » Thu Apr 20, 2006 9:46 pm

does the hairpin contain an inverted repeat (are you designing a miRNA)? Certain sequences and structure are very unstable regardless... and maybe lost or selectively degraded... (i don't know about sure2 competant cells.)

I think you should call the manufacturer actually.. and ask them to help you troubleshoot the problem. They'll be able to tell you what DNA sequences are compatible with that type of competant cells. (and they may be willing to refund money and/or select a better system for your project)

also... i might be able to refer you to some literature that you may be able to use to modify your construct. (.. I can' gurantee that it will help .. but it's just an idea)

I'll pubmed.. and look for the paper.. and get back to you tomorrow. ( call the manufac. in the mean-time)

Good luck!
- KIM

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