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Cloning long fragments - (Aug/03/2004 )

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hi there,

I have been trying to clone 3.4 kb insert into pcDNA3.1- for months without any success.

The insert is subcloned in TOPO from where I cut it out by KpnI and NotI and purify it by gelelectrophoresis using 0.5 % agarose and 1x TBE buffer.

I cut a small visible fragment out of the vector using the same enzymes and purify the vector in the same way as described above.

For gel-extraction I use the Qiagen Kit.

Ligation is done by T4-Ligase from NEB for 90 min at room temperature. The insert:vector ratio is about 3:1, total amount of insert DNA is about 300 ng.

To get rid of salts I perform microdialysis by using a millipore membrane on water for 20 min.

Afterwards I transform XL Blue MRF E. coli from Stratagene and the next day there is not a single clone on my ampicillin/agar-dish!

Could anyone imagine, what went wrong or is experienced in cloning fragments of larger size?

I have been trying for months now and I am kind of desperate!

Thanks for your help
claude

-claude-

Few questions..

Do you have a positive control in your transformation of E.Coli? So you know your bugs are competent and the procedure works

When ligating, do you have vector without insert? If the insert only stick to one end, the plasmid wont close, and you wont get any colonies. If you get lots of colonies with vector alone, one of enzymes haven't cut.

I don't think you need to get rid of salts before transformation. I usually use 5 ul of ligation reaction to 50 ul of bugs.

-Sprag-

I'm with Sprag, no need for salt removal unless you are electroporating. Have you checked to see you still have a good amount of vector after dialysis? I only ask because I've never tried that before.

-wirly-

Hi,
some possible steps you could check:
1)is your ligation working?
I would test ligase on some other DNA, eg. you could purify BOTH your vector and small insert that you normally discard (how big is it? Qiagen kits go down to about 70bp) and then religate. Then either run on gel to visualise (run with closed vector, and cut vector as controls to copmare against), or transform into bugs and check for colonies.
Also you can run a sample of your actual ligation reaction on gel, and if you get good ligated plasmid then transform the rest.
I do my ligations overnight at 4C, or even over a weekend, no harm in going for a longer time.
2)As Sprag said, is your transformation working?
I always do a control, eg. your initial vector, that should give you plenty of colonies. Vector without insert is a good negative control, but going by your enzymes the vector should not reclose on itself, plus you'd get colonies that way, so I don't think that's the problem.
3) Your dialysis step- I don't bother, perhaps you lose DNA there somehow? If you do it, check DNA concentration afterwards
4) Have you tried other ratios of vector to insert? Could help
5) Do you have bacterial control, ie is your amp concentration ok?
Good luck!

-kant0008-

QUOTE (kant0008 @ Aug 3 2004, 06:08 PM)
Hi,
some possible steps you could check:
1)is your ligation working?
I would test ligase on some other DNA, eg. you could purify BOTH your vector and small insert that you normally discard (how big is it? Qiagen kits go down to about 70bp) and then religate. Then either run on gel to visualise (run with closed vector, and cut vector as controls to copmare against), or transform into bugs and check for colonies.
Also you can run a sample of your actual ligation reaction on gel, and if you get good ligated plasmid then transform the rest.
I do my ligations overnight at 4C, or even over a weekend, no harm in going for a longer time.
2)As Sprag said, is your transformation working?
I always do a control, eg. your initial vector, that should give you plenty of colonies. Vector without insert is a good negative control, but going by your enzymes the vector should not reclose on itself, plus you'd get colonies that way, so I don't think that's the problem.
3) Your dialysis step- I don't bother, perhaps you lose DNA there somehow? If you do it, check DNA concentration afterwards
4) Have you tried other ratios of vector to insert? Could help
5) Do you have bacterial control, ie is your amp concentration ok?
Good luck!

So, I tried to use the the small fragment which is about 700bp as a control reaction and ordered new ligase, high conc., but the control showed only a single colony, as my main reaction didn't work and no colony appeared!
Could there be a problem in the transformation step? I am pretty sure that the E.coli are competent and that the ampicillin-conc. is allright.
Is it possibly the water ? I use bidest for all my work.
And what about the NZY+ broth? Might there be a problem with that?
I even exchanged TBE to TAE as I read, borate could inhibit the ligation.

-claude-

Hi again,
sounds like you ar ehaving "fun":(
Ok, transfrormation. I'd use your initial vector- don't cut and ligate, just the original plasmid, as the transformation control. If it doesn' give you good number of colonies you'll know that it's not the ligation and there is something wrong with the transformation.
If it is the transformation that doesn't work you can try making fresh competent cells, it may be the problem (how do you make them? I can give you an easy protocol). Double check the ampicillin conc just as an extra precaution.
I don't think water could harm your reaction in any way, it's only water.
Not sure about broth, I've not used it myself, but theoretically there shouldn't be anything in there that inhibits bacterial growth.
And what do you use TAE for? Is it in the Qiagen prep step? I use water, it works ok, just leave your tubes 5mins before spinning for the last time.
Good luck!

-kant0008-

Fail-free controls:

1) Ligation

Digest the plasmid you are using with a single enzyme, clean up with Qiagen and ligate. Use a lot of DNA. Run the ligation reaction product on a gel along with an unligated (linearised) sample of the plasmid. If the ligation product runs like a plasmid (ie gives 3 bands) then ligation was successful.

2) Transformation

Like kant0008 said, transfor your cells with just the original plasmid, you should get A LOT of colonies. If you don't get a lot, then your cells are not very competent. KEEP COMPETENT CELLS ON ICE ALL THE TIME, DURING PREPARATION AS WELL AS DURING USE. ALLOW CELLS TO THAW ON ICE. DO NOT VORTEX TO MIX THE DNA WITH THE COMPETENT CELLS. USE CUT TIPS.

Also, I would give at least 4 hours for the ligation reaction.

Good luck

-InvisibleSurfer-

Hello,

ad Transformation:

I transformed the uncut vector as control and got very, very many colonies on the plate as the other one was still empty. So my conclusion is, that the problem is not the transformation!

ad Ligation:

I performed this cutting and re-ligating of the plasmid. Running it on a gel along with linearized and original plasmid as controls, I found kind of a smear for the re-ligated plasmid, which did not have the typical plasmid-pattern but also differ from the single-cut plasmid.

Is there a problem with the vector itself?


Many thanks for your help

-claude-

hm, I don't think you have a problem with your vector, ligation reactions give a smear when run on a gel, could you see any indidual bands in the re-ligated lane and if so how many?

I think your ligation worked ok. Way I do cloning:

(steps 1&2 in parallel)
1. Digest vector with required enzymes and cut band out
2. Digest insert (PCR product or whatever) and cut band out
3. Mix digested vector & insert and purify (I use Qiagen)
4. Add ligase, buffers etc and pray

HOWEVER, I do not calculate the vector:insert ratios, so this might need a bit of tweaking. Also, make sure the DTT in the ligase buffer is resuspended 100%, put the tube under warm, running water from a tap if necessary. Hope this helps.

-InvisibleSurfer-

Hi!
No wonder you are frustrated!:) This sounds like a pain!
Hm, about your ligation on gel: what did you see except for the smear? You should see a lot of ligated plasmid (which runs somewhere at the top of the gel, higher than the plasmid size), you may also see some unligated plasmid at the plasmid size. As long as you see the ligated plasmid it should be ok.
Did you try your ligase on some unrelated DNA?
Running out of ideas:(
Good luck!

-kant0008-

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