How is the insert prepared?

Here's what I would do:

1. Make sure your enzymes are cutting at the expected places. Do some testcuts just to be sure (with one enzyme and another known enzyme or one enzyme at a time, etc). If unexpected bands are appearing, may be worth sequencing. It could be that one of your enzyme isn't cutting real well, hence no colony on the vector only control.

2. Increase vector amount. 50 ng of vector is usually enough but your vector is quite large and 50 ng may not be sufficient.

3. Try less CIP (AP). I usually just add ~0.5 uL of standard NEB CIP to my vector digest reaction, incubate at 37 deg for 15 min then run on gel.

Good luck! Sometimes that's pretty much you need in mo bio.

Ilya-

I PCR'd out my insert from a plasmid and added BstBI sites to the ends followed by TOPO cloning. I sequences the TOPO clones and found that there is an extra BstBI site that I didn't expect. This made it more difficult because I had to do a partial digest with different dilutions of enzyme followed by running it on a gel and excising/purifying the band of interest. This left me with a very small amount of DNA. The problem is that for this particular experiment there aren't any other cut sites that will work.

Joyce-

I believe the enzyme is cutting because I am getting a band at the correct M.W. and excising/purifying from gel.

I will try increasing the amount of vector.

What would less AP do?

I didn't run it on a gel after AP treatment because I had such a small amount of DNA from the initial gel purification I was afraid that it would go even lower if I ran it out again and purified.

Thanks!

I've done mutagenesis work with large plasmids (VGCC, VGSC) and I found that over-incubation with too much AP (alkaline phosphatase) can lead to inability to re-ligate (over dephosphorylation??). I'm actually not sure mechanistically why. I think as long as you get rid of the AP before ligation (either by heat inactivation and EtOH precip or by running on a gel then purify) you should be okay.

What are your ligation conditions?

Stephen-

20ul reaction

50ng vector

30ng insert (3:1 molar ratio)

Invitrogen T4 DNA ligase

1hr at RT, diluted the reaction to 100uL with ddH2O and used 2 uL for transformation of MAX efficiency DH5a cells.

Hi Joanna,

I would ligate overnight if I were you. You are not doing the easiest of ligations due to the large plasmid and insert size, so increasing the ligation time will help a lot. Often for overnight incubation, you can do it at 16C (which I often do).

Hope that helps - let us know if you have any luck.

Steve

I have a couple of ideas:

First, your uncut vector is 13kb but with insert it goes over 15kb which lowers transformation efficiency. Are you doing a heat transformation or electroporation? I believe electroporation is more suitable for large plasmids.

I also agree with Stephen about the overnight ligation at 16C. Maybe avoid diluting the reaction as well. The restriction enzyme you use leaves only 2 bp overhang, this is a good enough reason to go for overnight ligation.

Ilya.

Hi Joanna,

as Stephen already mentioned your ligation is very tricky. There are several points to consider:

1st: What kind of restriction enzyme are you using? Does is have a 4base "sticky-end" overhang or is it blunt-ended (I hope not)? If you use sticky ends, annealing you plasmid and insert could help a lot. Therefor mix vector and insert at 1:3 molar ratio and heat to 42 degrees celsius briefly. Then just turn of the heating block, so that it cools down slowly to room temperature. Afterwards add ligation buffer and Ligase (If it is quick ligase stick to protocol of supplier!). For normal T4 Ligase too long incubation can also destroy your plasmid.

2nd: DH5alpha might not be the right E.coli strain to propagate plasmids as big as your final construct. When I was cloning some big vectors >10kb I usually used E.coli HB 101. But there are several other E.coli strains working well for big plasmids.

3rd. You should try to do you AP treatment as short as possible, or even omit it, since prolonged AP treatment destroys your overhangs, resulting in non ligateable plasmid.

4th: AVOID any exposure to UV!!!! You mentioned that your vector was gel purified. Use some kind of RedSafe/GelRed or similar and a blue light table instead. Any exposure of your vector to UV results in TT dimers which will bend/kink the DNA and decrease the chance of ligation.

If everything does not help, In-fusion cloning could be an alternative. It isn't cheap, but always worked for me when clong big vectors/inserts.

Best Kai

Hi, I once cloned constructs of up to around 19 kb, but this was quite tedious - extremely low efficiency. You may change your ratio - if I remember correctly, I used a ratio of 1:10 - but had to double-check for multiple inserts. I also tried many different batches of competent bacteria...

Hi Joanna!

Was the extra-BstB1 site introduced erroneously during the PCR amplification or was it present in the original sequence? If it is an artifact of the PCR, then redo your amplification using a high-fidelity polymerase and digest directly the fragment. Sequence your insert on the final plasmid.

I think you have very good suggestions above such as you should respect the molar ratio between plasmid and insert (it doesn’t look that you do), etc... It is true; recheck if your E.coi strain is competent to harbor big plasmid. Don’t dilute your ligation reaction, just transform with 1 or 2 ul.

It is not necessary to de-phosphorylate your plasmid when the ligation engages cohesive ends. So, you can skip completely the AP treatment step. The ligase we use in cloning needs a 5’ phospho-end to perform ligation to a 3’-OH end. Normally, the background from re-ligated plasmid is low. Check your transformation by colony-PCR.

Also, try another ligase. Expensive doesn’t always mean the best

Thanks, Joanna, for your private message. Yes, I used much more insert, but I know there are many, many reasons to stick to published protocols and ratios, so you should try other things first (better, freshly made super-competent bacteria). I was in Germany in a highly competitive (and generally hostile) group at the genetics institute of the nuclear research center in Karlsruhe - with another two competing postdocs and one stealing phd student around in the same lab working on the same project, i.e. my isogenic lambda-phage clone and restriction map with all useful restriction sites mapped and then sublconing fragments for creating CD44 partial knock-out constructs - and then not even getting on one paper of that work which helped so many.... but that was typical for those questionable professors - getting German awards. After three years I was at least 6 months ahead of anybody else and, to the best of my knowledge, had the first complete two K.O. constrtucts in the world of about 19 kb on an isogenic background for ES-cells. Nevertheless, I learned, that using freshly made Dh5alpha bacterial cells were best, but each preparation was differently successful, so we ended up having one large batch of super-competent cells aliquoted and frozen which helped a lot . I learned to make 100 -300 mini-/midi-preps a day for months and analyzing them in parallel to continue fürther cloning, sometimes it needed 300 preps to get one successful clone... hope your cloning will be easier.

Joanna- don't overthink this problem. Ligations only fail for one of three reasons. First, your DNA ends are not compatible, Second, you have a chemical inhibitor or damaged DNA (e.g. excess UV treatment) that blocks successful ligation. Third, your vector has high background (incomplete digestion), and you've already ruled this option out. I suspect reason #2, especially since you indicated your DNA was of low quality so you had to precipitate it.

First, try a control ligation with insert alone, and check for a migration shift on a gel. If this fails, you insert is no good (no sticky ends, or contaminated with a chemical inhibitor). A common problem is that some restriction enzymes don't cut near the ends of PCR amplified DNA unless you include 3-4bp of flanking 5' sequence. If your insert is ligation-competent, then you need to re-check your vector. Maybe it didn't digest with both restriction enzymes (a common problem in closely spaced MCS), or perhaps you over-treated with CIP (CIP contains contaminating exonuclease activity that can chew off your sticky ends if you treat with too much enzyme or for too long).

If none of this helps, please update your post with details about which restriction enzymes you are using and your methods of insert preparation and gel isolation.

 Dr. Kai F. Albring ,

Will you explaine why you sujested to heat up the ligation reaction to 42 degree?

Especially if dealing with blunt end sites.

Thanks

Dear Imam,

this "annealing step" is usually important when dealing with sticky ends. It denatures the annealed plasmids and annealed inserts, so they are available for annealing with each other. I actually wasn't aware that it also improves blunt end ligations.

If you want to improve your cloning in the future, you might not even need to use ligases any more. Have a look at the publication of Koskela et al.

http://www.ncbi.nlm.nih.gov/pubmed/25370826

I tried it and it works perfectly.

Happy new year,

Kai

Add fresh ATP to a final concentration of 1mM. This will help if you have frozen and thawed the ligation buffer too many times.