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problems in cloning, need help


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#1 dan_

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Posted 26 May 2010 - 07:27 AM

Hi everyone,

I try to cloned a 2.8Kb insert, cut AflII-DraIII into a 14kB plasmid, also cut AflII-DraIII for 4 months, without succes.

I have put my ligation mixture on gel, and I can see a shift between my band on my ligation mixture compared to my linearise vector, so I think that my problems don't occured during the ligation step, but after, during the transformation or the selection of the clone on plate.

I have checked the bacteria (SUREcells): they are competents. I did a ligation control with pCR2.1 vector cut with EcoRV and I got about 1000 colonies. I did a ligation with only the vector without insert and I got no colony on plate. I did a transformation with the uncut vector and I got few colonies (about 50) on plate.

So, I'm thinking that the problem is that the vector, because of it 16kb long, is instable in bacteria.

This is the way I proceed to get clone on plate. Transformation of my DNA (10uL) in SUREcells bacteria (100uL), heat sock at 42C 90 sec, add medium without antibotic (1ml) and they grow 90 min at 37C with gently shacking. Then, I spin cells, resuspend in 100 uL of LB medium and plate them on LB-ampi plate. Plates are incubate o/n at 37C. Usually, I get 20 colonies on each plate. Them, I pick up several clone in 3 ml of LB-ampi and they grow at 30C for 16-20 hours without shacking. Following miniprep, all clone have plasmid DNA, but no one have the same uncut patterns, compared to my uncut vector (16,8 kB long).

So, what do I do wrong? Do you have suggestion that can help me? Does it exist an other strategie? I'm at a point that I am ready to try anything, if it could give my good results.

Thanks in advance,

dan_

Edited by dan_, 26 May 2010 - 07:29 AM.


#2 TomH

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Posted 26 May 2010 - 08:36 AM

Why do you grow your cultures for minipreps at 30oC with no shaking? Is it a strain specific thing? I usually grow mine at 37oC with shaking to get the most growth and most DNA.

Where are you cutting your vector to assesses if ligation is successful? Have you tried cutting with two enzymes, one with a site in the insert and one with a site in the vector or simply with AflII and DraIII? That way you would cut out a product that would only be visible if you had an insert.

What do you see after miniprep? It's unclear what you mean by 'but no one have the same uncut patterns, compared to my uncut vector (16,8 kB long)' as surely you'd expect a successfully ligated product to have a different pattern to your empty vector?

#3 dan_

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Posted 26 May 2010 - 10:26 AM

Thanks TomH for your reply!

I grow bacteria at 30C with no shaking because recombinaison event in bacteria may occur when you grow clones at 37C. With or with no shaking do not make any difference for me, instead that I get less DNA per prep, but I really don't care, I have enough DNA to analyse it on gel and to sequence it.<

The way I analyse DNA to see if I get good clone it that I put uncut DNA coming from my prep on gel (with supercoiled DNA Ladder, invitrogen and with uncut vector for positive control). This is a faster and cheaper way to analyse DNA instead to digest DNA with restriction enzyme and look at the restriction profile on gel. For all plasmid that I work with, It gave me good results. In this case, if I run the gel enough, I should be able to see difference between uncut vector ligated with imself (14kB) and 16.8 kB DNA (vector + insert). But, every time, I get DNA ranging from 8 to 14 kB and I don't know what happens.

dan_

#4 phage434

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Posted 26 May 2010 - 10:51 AM

The two hours it takes to cut your plasmid with a restriction enzyme is trivial compared to your four months. Start doing digests. The gels are much much easier to interpret.

It sounds as if your main trouble is getting good transformation of your parent plasmid. A shift to electroporation would probably help.

You should be shaking your cultures at 30C. They will grow faster, and be healthier. Why are you doing things differently from common practice, especially if you are having troubles? If you are worried about stability, your cell recovery and colony growth should be at 30C also.

Details on your cutting and religation protocols might reveal issues. Let us know how you are doing it.

#5 pDNA

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Posted 26 May 2010 - 12:59 PM

AflII seems to be a very tricky enzyme ...and not very reliable refering to what i have read at the NEB homepage.
Its inhibited by salt concentrations >50mM and it is stated that the ends ligate poorly. NEB suggests blunting the ends to increase cloning efficiency. (one should be alarmed if a company warns of low ligation efficiency!). Maybe you can try blunting the ends as suggested? ...or switch to another cloning strategy using reliable enzymes.

Additionaly i would also recommend to do restriction digests of your plasmids ...this is state-of-the-art and gives you essential information on your experiment!

Concerning the culture for minipreps:
Please incubate them shaking (like anyone does!) ...E. coli loves oxygen and starts doing wicked things when oxygen-limited (plasmid instability, inductions of transposons, increased recombination events, just to name a few). Just think about a bioprocess using E. coli ...what is the main issue cultering E. coli in large scale? Aeration of the culture is vital for a successful bioprocess (sometimes pure oxygen is used to provide enough oxygen) ...and this priniciple should also be applied for lab scale cultivations. Think about that nex time!

Good luck!

Regards,
p




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