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plasmid re-ligation problem

plasmid re-ligation problem - Protocols and Methods Forum

plasmid re-ligation problem - Post Any Protocol, Method, Technique, Procedure or Tips / Troubleshooting for any Molecular Biology Technique.


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  #1  
Old 08-11-2004, 06:48 PM
Vagelis
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Default plasmid re-ligation problem



Hello everyone,

I am trying to construct a recombinant pSP189 vector that is going to
be used for future site-specific adduct studies. In brief, the parent
vector is digested with the XhoI and BamH I restriction enzymes (from
NEB). Both enzymes are single cutters and produce incompatible sticky
ends, and the distance between the two restriction sites is 114bp. We
want to ligate back to the double-digested vector a modified,
synthesized ds114mer.

However, I encounter a problem during the cloning procedure; the
double-digested vector re-ligates back to itself, even though the two
ends are incompatible.

The parent plasmid is digested separately with either the XhoI or BamH
I enzymes according to the following protocol:

- 10ug pSP189 plasmid (1ug/ul)
- 10ul BamH I or NEB 2 Buffer
- 1ul BSA
- 100 NEB units of BamH I or XhoI
- up to 100ul with ddH2O
- 2 hours, 37C

The single-digested plasmid is purified on a 1% agarose gel (overnight
40 volts).
The enzymes appear to cut efficiently, when used separately, as is
evident from direct DNA sequencing of the plasmid and also from the
agarose gel photos

The gel slices containing the single-cut DNA are excised from the gel
and the DNA is purified with the QIAquick gel extraction kit (QIAGEN).
Then, the second digestion is performed (same protocol) and the
double-digested plasmid is purified again on a 1% agarose gel
(overnight 40 volts). The plasmid DNA is recovered again with the
QIAGEN kit and then it is used for the following ligation reaction:

- 1ug double-digested plasmid
- 1ul (400 Cohesive End Ligation Units) T4 DNA ligase (NEB)
- 5ul T4 DNA ligase buffer
- up to 20ul with ddH2O
- 2 hours room temperature

The ligated product is loaded on a 1% agarose gel (overnight 40
volts). The double-digested vector re-ligates proportionally to the
amount of T4 DNA ligase. In fact, virtually all of it re-ligates in
the presence of 4000 units of T4 ligase. Also, when the modified
114mer is added to the ligation reaction in high concentrations, it
tends to inhibit the re-circularisation of the vector.

The same problem occurs even when I perform double digestions or when
I do not purify the digested vector with agarose electrophoresis. I
also dismissed the possibility of contamination in my ligation buffer
or in the T4 ligase.

My guess is that there is either a misdigestion problem during the
second digestion or that the 114mer is indeed excised but not
separated from the rest of the vector.

Do you have any suggestions on how to overcome this problem?
Thank you in advance,

Liapis Evagelos
PhD student,
Biocentre
Leicester University
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  #2  
Old 08-11-2004, 07:35 PM
Ferdinando Pucci
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Default plasmid re-ligation problem

On 11 Aug 2004 11:48:01 -0700
[Only registered users see links. ] (Vagelis) wrote:


How did you realize that is the vector recircularized? Did you see a
band a little under the supercoiled wt vector or you have another
plasmid the same size?


I'm not a PhD nor a MD but i'm working with this to get a degree, so i
can try to say something not so strange :-) imho probably there is a
degradation problem of your stickies that become blunt and... I would
try to change the purification kit or to have more care with hazardous
passages.

Good work!

PS: i'm used with so much RE as 1U/ug of DNA but you beat me! :-)
PPS: maybe with a conc of 50ng/ul in the ligation reaction there could
be too much probability of self ligation, you should calculate it
(search previous posts here)
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  #3  
Old 08-12-2004, 04:48 AM
Austin P. So (Hae Jin)
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Default plasmid re-ligation problem

Vagelis wrote:

You have to remember that you are not ligating one molecule of plasmid,
you are ligating trillions of them, so you will get end to end ligations
occurring:

A---B

A---BB---AA---B...etc...

especially at the concentration of plasmid (1 ug in 20 ul) you are using.

Use much less plasmid (25 ng), and then add 2:1 molar ratio of insert to
plasmid for your ligation at the volume of 20 ul.

That should be more than enough for your transformations.



Of course it is going to...and that is what you want, isn't it?

If you want to be absolutely sure about your ligation, treat your doubly
digested plasmid with phosphatase so that it cannot self-ligate
(circularize or concatemerize).

You can relax...there is nothing going "wrong" and there is no
"contamination"...

Good luck

Austin

BTW...You are overkilling on your purifications...the Qiagen
purification kit should be more than enough.

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  #4  
Old 06-09-2009, 11:25 AM
Pipette Filler
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Default Re: plasmid re-ligation problem

It worries me you leave your tank on overnight!! I just set mine to 100v and run it for 20-30 mins.
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  #5  
Old 06-20-2012, 08:29 PM
Pipette Filler
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Default SAP treatment of plasmids

Hi
do you know maybe any other methods of purifying plasmid vector after SAP treatment than phenol chloroform ? maybe columns???
txc
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