6 replies to this topic

[Scuffle]

Another poor soul having problems ligating.

I want to insert a 1k insert into a 13K plasmid.

I amplified the insert with primers that added an xba1 site to the 5' end and a kpn1 site to the 3' end, and got a nice strong band after PCR. I cleaned this up with a Qiagen PCR cleanup kit, and then performed the double digest. Kpn1 can't be heat inactivated, so I used the PCR cleanup kit to purify again.

I digested the plasmid with the same two enzymes, and ran some on a gel alongside uncut vector to confirm that it's been cut. Since it's too big for the Qia kit I cleaned it up this reaction by phenol chloroform followed by spin-dialysis. I checked on a gel and still have a decent concentration of cut vector.

I tried to spec check, but got some odd readings for the vector - much too high, especially considering the moderate strength band I observed on the gel.

Because of this, in a 10ul ligation reaction with t4 ligase, I simply used 1ul of vector and three of insert. I let it ligate for 4 hours, then transformed 4ul into XL1 blue and DH5alpha electrocompetent cells (since I sometimes get better results with one or the other) and plated on selective plates. No colonies.

Any ideas where I should start to work out what's gone wrong?

Edited by Scuffle, 02 July 2010 - 01:44 AM.

[HomeBrew]

Hi Scuffle -- welcome to the BioForums!

Scuffle, on Jul 2 2010, 05:42 AM, said:

Any ideas where I should start to work out what's gone wrong?

I have a few...

Scuffle, on Jul 2 2010, 05:42 AM, said:

I amplified the insert with primers that added an xba1 site to the 5' end and a kpn1 site to the 3' end...

How many extra bases did you add 5' to your restriction sites? See here.

Scuffle, on Jul 2 2010, 05:42 AM, said:

I cleaned this up with a Qiagen PCR cleanup kit, and then performed the double digest. Kpn1 can't be heat inactivated, so I used the PCR cleanup kit to purify again.

Do the PCR, then the double digest, then gel purify once.

Scuffle, on Jul 2 2010, 05:42 AM, said:

Since [the vector is] too big for the Qia kit I cleaned up this reaction by phenol chloroform followed by spin-dialysis.

Use the Qiagen kit to purify your vector.  Modify the protocol by adding one volume of isopropanol and one volume of distilled water to the solubilized gel slice.  Elute in 65C EB buffer or water.  I have recovered fragments as large as yours using this method.

Scuffle, on Jul 2 2010, 05:42 AM, said:

I tried to spec check, but got some odd readings for the vector - much too high, especially considering the moderate strength band I observed on the gel.

This is likely due to phenol contamination in your sample.  Use the Qiagen kit as above to avoid this.

Scuffle, on Jul 2 2010, 05:42 AM, said:

I let it ligate for 4 hours, then transformed 4ul into XL1 blue and DH5alpha electrocompetent cells (since I sometimes get better results with one or the other) and plated on selective plates.

Let the ligation reaction proceed at 16C overnight, and transform chemically competent cells.

In addition, add 0.28 g/L (~1 mM) guanosine (e.g. Sigma G6752, FW 283.24) as a UV protectant to the 1× TAE used to cast the gel and as running buffer during electrophoresis.

If you still can't get clones, I would first suspect that your PCR product is incompletely digested.  Capture your PCR product in a TA cloning vector, and transform E. coli.  Do a plasmid mini-prep on a culture of this clone, liberate your insert from the TA cloning vector with  XbaI and KpnI, and recover your fragment via Qiagen's gel purification kit.

[Scuffle]

Hi! Thanks for the welcome and the thorough reply. Even through I've only just joined, this forum has been helping me for quite a while since posts often come when I Google search for help with my experiments.

My primers added a 4 base extension  (sorry I should have mentioned that) so hopefully that's not the problem. Why do you recommend gel extraction of the RE digested PCR product? I was hoping a Qia PCR purification would be sufficient to flush out the REs and the cut-off ends from the PCR product - I like to avoid gel extraction where reasonably possible.

I was hoping that spin dialysis would remove any contamination from the phenol-chloroform, but I've been a bit dubious about the protocol we use in the lab for a while, so I'm not suprised if it's not doing what it should. I'll try Gel Extraction with your modification and see what happens. Although, we don't have any heat competent cells at the moment, so I'll have to stick with the cells I have.

Thanks for the tip about TA cloning the PCR product, too.

[HomeBrew]

Scuffle, on Jul 2 2010, 08:27 AM, said:

Why do you recommend gel extraction of the RE digested PCR product? I was hoping a Qia PCR purification would be sufficient to flush out the REs and the cut-off ends from the PCR product - I like to avoid gel extraction where reasonably possible.

I gel purify everything when cloning -- I just digest my vector and insert at the same time and recover them from the same gel.  Sure, spin columns are easier and quicker, but I've learned from long experience that the time you save doing it that way is not worth it -- too many failed ligations, causing me to have to repeat the entire experiment.  With everything gel purified, my cloning experiments more often than not work the first time.

[chromatin]

Potential problems I can think of:
1. How do you know both of your enzyme cut both inert and plasmid. If size of the DNA is not changing enough you cannot see it on the gel. You think both of them cut. You may want to try TA subcloning or similar strategy first, then cut with enzymes. For example you can try to clone PCR product into TOPO vector.

2. I would use Qiagen columns. Size is not a problem. You can purify much bigger DNA with these columns. Efficiency may change but it's not a problem since you need only little bit DNA. Your DNA might be contaminated by chloroform. Your high reading is coming from chloroform. If you carefully look at full spec, I bet you will see the peak absorbance around 270nm not at 260nm

3. If you are using gel purified DNA to spec often you will get wrong absorbance values. I know very well that after buffer QG purification you have contamination coming from QG/agarose. Your OD260 will be wrong. You can only tell this if you get a full spectrum of your DNA, 260nm to 800nm. Then you should able to tell how clean your DNA is. If you do not know how to read spectrum, then I suggest you determine the concentration of DNA by gel.

Good luck,
http://www.bioprotocols.info

[HomeBrew]

chromatin, on Jul 3 2010, 10:42 AM, said:

3. If you are using gel purified DNA to spec often you will get wrong absorbance values. I know very well that after buffer QG purification you have contamination coming from QG/agarose. Your OD260 will be wrong. You can only tell this if you get a full spectrum of your DNA, 260nm to 800nm. Then you should able to tell how clean your DNA is. If you do not know how to read spectrum, then I suggest you determine the concentration of DNA by gel.

This may very well be true, but I never bother to spec my DNA samples anyway -- I just estimate relative concentrations from the EtBr gel, combine, and go (another advantage to gel-purifying both the vector and insert on the same gel)...

[Scuffle]

I gel extracted the vector, and seemed to get an OK yield - thanks for your suggestions. And thanks for the info about specing, Chromatin.

Still no luck with my ligation, however. I tested that the vector can be cut with both enzymes individually last week, and that's fine, so I'll try TA cloning my PCR product and see what happens.

Edited by Scuffle, 07 July 2010 - 04:54 AM.