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Molecular Cloning Forum DNA cloning forum. Discuss the cloning of plasmids, vectors, DNA, RNA, cDNA, proteins, and other molecules for expression, sub-cloning, and other methods. Also discuss digestion, ligation, transformation, plasmid preparation, cDNA library screening, and competent cells here.


REALLY Desperate!! - simple ligation not working!

REALLY Desperate!! - simple ligation not working! - Molecular Cloning Forum

REALLY Desperate!! - simple ligation not working! - DNA cloning forum. Discuss the cloning of plasmids, vectors, DNA, RNA, cDNA, proteins, and other molecules for expression, sub-cloning, and other methods. Also discuss digestion, ligation, transformation, plasmid preparation, cDNA library screening, and competent cells here.


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  #1  
Old 05-12-2011, 01:53 PM
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Exclamation REALLY Desperate!! - simple ligation not working!



Hello,

I have been trying to make a very simple construct for many months and can't find out where the problem is! If it doesn't work soon I don't know what I'll do...

Here is what I've tried:

Vector DNA: 7 kb : Single digest with Age I, gel purify.

Insert: 1.4 kb PCR product, PCRd with primers that have AgeI linkers added (5 bp overhang on each end)

Protocol: Digested vector and insert with Age I. CIPd vector and gel purified. Gel purified insert.

Ligated at 2-3:1 molar ratio of I:V in 10 ul with 40 ng vector. Transformed 5 ul into 100 ul NEBalpha cells.

Results: Same number or less colonies on V+I as V.

This has been repeated many times, with the following variations:

Age I Enzyme tried:

Promega Age I still had some uncut but ran gel a long way.
Biolabs Age I still had a small amount uncut.
FastDigest Age I did not see any uncut after digestion. (Used at least 5x excess of enzyme and digested up to 4 hours)

CIP tried: Promega, Roche (gel purified after; did not heat inactivate)

Ligase tried: Promega, Biolabs. Ligations carried out 16 C overnight.

For the insert, I also put the PCR product into the Zero Blunt vector and cut it out with Age I and gel purified the 1.4 kb band so I know the insert was successfully cut.

DNA fragment purification methods tried:
Qiagen gel and PCR kits (eluted with H2O);
Roche glassmilk (gels) and PCR cleanup columns (eluted with TE or Tris pH 8)

Also bought in some BioRad Mol Biol grade agarose for gels and HPLC water to use for enzymatic reactions.

I also found a different prep of the vector I had made in a dam- host and tried that.

Transformation results:

V only - quite a few colonies (a couple hundred colonies on a 10 ul of 100 spread onto plate)

I only - no colonies

V+I same number of colonies as V only

I always get Vector only background and nothing else. The Ampicillin is working because when I transformed a control construct I got a much higher no of colonies.

I think there is a problem ligating the V+I but have no idea how to troubleshoot this any further.

Is there any chance my competent cells dont like the 1.4 kb fluorescent tag Im putting in? The tag is Green Cherry. The construct Im trying to drop it into has been used successfully before to make other tagged versions like mCherry.

Someone....please.....help.

Thanks!
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Old 05-12-2011, 04:12 PM
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Default Re: REALLY Desperate!! - simple ligation not working!

One thing I haven't been doing is heat-inactivating the T4 DNA ligase prior to transformation. But this is something I haven't always done in the past and not had any problems before. Could this be significant? I am doing 10 ul ligations with 1 ul of ligase in 10 ul final volume, then transforming 5 ul of that into 100 ul of NEB5-alpha competent cells.
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Old 06-09-2011, 02:24 AM
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Default Re: REALLY Desperate!! - simple ligation not working!

Vector digestion is a problem for you,in that you are having undigested vector,that is very much dominating in the transformation always you will get undigested vector.even i had this problem.For digestion take 1-2ug vector DNA more enzyme and keep for long time(2-4hrs) and run undigested and digested vector side by side.run at 50volts for long time till dye front run out.then cut the band for gel elution then proceed ligation.this will help you.best of luck.
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Old 07-12-2011, 10:05 AM
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Default Re: REALLY Desperate!! - simple ligation not working!

Hi dsDNA,
I think there are different levels to this problem. I want to tell you that simple 1.4kb insert (fluorescent or not) has no effect on bacterial cells. So remove this as your reason of things not working. I will mention some imp reasons you can check if they will help you:
1. Resynthesis primers: Sometimes these primers are not full length so they amplify different stuffs.
2. Use nuclease free water.
3. Use freshly prepare agarose gel.
4. Check ur PCR amplified insert using some internal REs.
5. Do use high wavelength (low frequency) UV light to cut your insert out from the gel to minimize the DNA damage.
6. Use extra ATP in the ligation buffer (if its sticky end) and NO ATP if its blunt end.
7. Buy commercial competent cells and then try with them.
8. Age I could show "star activity" for longer incubation.
9. Use phosphatase for 15min at 37C to dephosphorylate the Vector only.
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Old 07-16-2011, 11:45 AM
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Default Re: REALLY Desperate!! - simple ligation not working!

If you get roughly the same number of colonies in Vector alone as in Vector+Insert, you either have uncut vector, or the CIP treatment wasn't efficient.

Also you're getting tons of colonies in the Vector only condition (100s of colonies from 10% of the transformation is WAY too many). This number should be almost zero normally.

Since you gel purify the AgeI digested vector, I expect it is probably digested. For CIP-ing use about 1ul of enzyme per ug of DNA (I use the CIP from NEB so concentration might vary for your suppliers).

Usually I do CIPing like this:
1. Digest vector with Restriction enzyme for about 1 hour at 37C. This will generate free 5' phosphates when the plasmid is cut.

2. Add enough CIP for the amount of DNA present. Continue to incubate at 37C for AT LEAST 2 more hours. This allows the enzyme to keep cutting and leads to dephosphorylation.

3. Gel purify.

If dephosphorylation was efficient you should have almost Zero colonies in your Vector alone control.

I expect this is the source of your problem. Re-digest / CIP your vector. You can even test it without using insert in the first instance. You should get very few colonies. If you plate out 10% of your transformation and you're getting more than 5-10 colonies from the vector alone, the vector preparation is no good and the cloning will not work.
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Old 05-09-2012, 05:53 PM
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Default Re: REALLY Desperate!! - simple ligation not working!

Vector seems to be the problem, since you are digesting subcloned insert and gel purifying it. Apart from the basic things already mentioned (minimise exposure to UV etc), you could try a trick to get rid of uncut plasmid using DpnI. To do this, you would need to PCR amplify the vector itself, which is not a problem with enzymes like Phusion. Design primers that contain your restriction site, plus 6 guardian nucleotides, directed the opposite ways on the plasmid. Run a long PCR (allow sufficient time for extension). This will result in a long linear PCR product identical to a cut vector, plus overhangs. Digest it just like you digest the insert, but add DpnI to destroy the original vector. Since the PCR product is not methylated, it will not be digested by DpnI. Gel purify and ligate with the insert you already have. Your background should go down to zero.
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