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Cloning long fragments


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16 replies to this topic

#1 claude

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Posted 03 August 2004 - 03:36 AM

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

#2 Sprag

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Posted 03 August 2004 - 08:03 AM

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.

#3 wirly

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Posted 03 August 2004 - 03:01 PM

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.

#4 kant0008

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Posted 03 August 2004 - 05:08 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!

#5 claude

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Posted 06 August 2004 - 12:16 AM

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.

#6 kant0008

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Posted 08 August 2004 - 05:22 PM

Hi again,
sounds like you ar ehaving "fun":wacko:
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!

#7 InvisibleSurfer

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Posted 09 August 2004 - 03:28 AM

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

#8 claude

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Posted 11 August 2004 - 01:43 AM

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

#9 InvisibleSurfer

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Posted 11 August 2004 - 03:04 AM

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.

#10 kant0008

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Posted 11 August 2004 - 06:20 PM

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!

#11 claude

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Posted 12 August 2004 - 01:21 AM

Honest, this really gives me physical pain!

On the Ligase-Function-Test-Gel I had three lanes:

1. single cut plasmid: on defined band of the proper size
2. original, untouched, virgin plasmid: 5 bands of all kinds in size (typical pattern)
3. cut and re-ligated plasmid: slight highmolecular smear, but no clearly shaped bands


I also tried to ligate DNA-Marker as another control and got the same. The single bands were gone and a slight smear was now visible on the gel.

Therefore I assume the Ligase is working properly.

I will keep on trying!

Thank you so much!

#12 InvisibleSurfer

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Posted 12 August 2004 - 01:58 AM

I think ligation has worked, try it now with your insert+vector, incubate for at least 4h at 4'C (preferably overnight at room temp), transform the next morning and good luck!

#13 sinni_2k

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Posted 27 September 2004 - 05:15 AM

Hi there!
I have the same problem at the moment. Have you ever thought of your cloning product being toxic for the bacs?
Anyhow i am trying to ligate a 2.5kb insert into pcDNA3.1 (NotI und HindIII) and I also get no colonies.
So, if you have found the key to successfull cloning with this plasmid, please let me know.....

best wishes for your work

sinni_2k

#14 Heini

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Posted 14 January 2005 - 07:18 AM

Hi Claude, Sinni_2k and others!
Have you had any success? I'm having similar problems and I'm going mad with my clonig.
I'm trying to clone 3,8kb PCR product to KpnI/BglII of the 7,5kb vector. I have also tried HindIII/HindIII instead of kpn/bgl.
I have tried many control ligations- nothing works!!! I have positive control(the intact vector) in transformation, which is ok.
I've used Qiagen to extract my fragments from the gel, but now I also tried Roche. - No colonies:(

I tried another plasmid, which had KpnI/BglII sitse (pBLCAT2), digested it and gelpurified the bands 2,8kb and 1,7kb, and religated this. NOTHING!!!
I have tried different ligation temperatures, times, DNA amounts in transformation (+inactivation)...
I tought that there is something wrong with the gel extraction, but when I changed to Roche it still didn't work.


PLEASE tell me if you had any success.

Heini

Edited by Heini, 28 February 2005 - 02:02 AM.


#15 claude

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Posted 28 January 2005 - 05:56 AM

Hello Heini,
i can really imagine you feel desperate! What I did then was, to make a really long lasting ligation reaction. At 4 degrees during the weekend. The next time I tried to transform, it worked! To be honest, I had two colonies on the agardish and was lucky to find one positive! But all the cloning I was doing the same way afterwards, was successful! I personally think molecular biology is kind of Voodoo. You never know, whatīs gonna happen!
So, the only thing I changed, was the ligation time.
I think, itīs worth to try.
Good luck
Claude




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