No ligation! Can't understand why! - (Mar/23/2008 )

Hello everybody,

I am currently trying to ligate a ~80 bp duplex synthetic oligo (that has the 2 required restiction site over hangs) into a double digested 5.2 Kb plasmid vector using NheI and SacI. So far I have tried:
1. 3 different T4 ligases!
2. Postive control that I know works (I have used the same vector and different insert (~35 bp duplex) that I had cloned before).
3. After restriction digestion of the plasmid, I have tried both gel purification and without gel purification (tried eliminating the ~60 bp unwanted sequence by Microcon spin down; which should eliminate anything less than 200 bp according to the product catalog)
4. Apart from the 3x molar conc of the insert, I have also tried 10x insert to vector ratio after reading one of the earlier posts...

Even after all that, I have not gotten a single colony with my insert ! Also, The synthetic oligo sequence has passed the HPLC quality control of the company and so, I think it is probably not the insert sequence issue.

I have spent about 2 months trying to do all this! I am completely bewildered and not sure what my next step should be...

I would appreciate any thoughts/suggestions that you may have.

Thanks in advance,
Micron

You have either ordered 5' phosphate on your oligos, or have treated them with PNK, right? Otherwise, they won't clone (well, they might if you have not dephosphorylated your vector). Are you certain the vector is being cut by both enzymes?

Why don'y just amplify your vector using two primers using PCR and then self ligate the product. You need high effeciency polymerase and 5' phosphate on your primer.

Thanks for both your replies.

Phage, I don't dephosphorylate my vector as the the 2 restriction sites are not going to ligate to each other (or atleast theoretically!). We don't need to phosphorylate the insert if the vector is not dephosphorylated. I have done it and I know this works. However, to rule that out, I used PNK to phosphorylate my insert and it still does not ligate and produce any colonies

Anwar, I am not quite following what you are suggesting....can you please explain it again?

Once again, thanks guys....

At my wits end....

If you find out the answer, please let us/me know. I also tried everything in the book and out of it and ive been having the same problem now for 6 months...though i recently got a a lot of colonies by using 20:1 vector:insert molar. Still dont know if theyre the right thing or not, double size so must be double ligated vector, but you never know! Good luck!

Can the oligos form secondary structures that will interfere with ligation? I am trying to fish for reasons why I would not get any inserts....

One more shot at ligation today and if I still don't get any colonies, I am going to look at commercial ligation. I called up a company and they charge $600 for subcloning the insert!!! I am trying to find something cheaper than that...has anyone used such resources that you can recommend? I am sure that my PI is not going to be too thrilled!

Thanks Gorkin, did you try sequencing your 20:1 insert plasmids?

= height of frustration
Micron

Gorkin, you mean 20:1 insert:vector ratio right? Just trying to make sure that I have tried everything that I possibly can before giving up hope!

Thanks,
Micron

Had a post-doc in my lab try to subclone in an insert for months with no success/colonies. He forgot his plates in the incubator for 24 hours (two day total incubation) and viola! Colonies with insert. It might be worth it just to throw your plates back in the incubator for another night. Nothing to really loose right?

I also hate to point out that you are using really crappy enzymes. Check out the NEB catalog for "cleavage close to the end of DNA fragments". For NheI, you MUST have at least 3 overhang bases to get 50% of your insert to digest after 20hour digestion. With a 2 hour digestion it's only 10%. With fewer overhang bases it's only 10-25% with a 20hour digest.

As for SacI, no matter what (overhang bases or length of digest) it only digests 10% of the insert!!!!

So you need one insert that was cut by both of these enzymes which, with only 10% getting SacI and maximum 50% (only if you have the overhang bases and digest for 20hours) for NheI. Otherwise it's about 10% for NheI. The odds are against you here. Sounds like you are going to have to do an overnight digestion just to have hope. How long are your digests currently?

Thanks rkay447, your reply has made me curious. I will throw the plates back in the incubator, as you say I don't have anything to lose! Though, I always thought that after 14-16 hrs all you got was satellite conlonies growing bigger. And, I have never understood what satellite colonies are and where they come from!

To answer your questions, I am not digesting my inserts. I had them synthesized with the right overhangs so that they will ligate to the digested vector. I have tried different times for the vector digestion, mostly for over 2 hrs and once overnight (to rule out the vector digestion issue). I have run the digested vector on the gel, and they look good. Also, I tried transforming the digested vectors (just to make sure that vector is digested successfully) and they don't transform. So, I know for sure that the vector digestion is successful.

Thanks everybody for your replies....

Still searching for answers ...

It was 20:1 and havent yet got the results from sequencing. Hopefully theyre ok! I really hope so...otherwise im in big trouble. Im still gonna keep trying as this stuff is supposed to be easy and I think its just because im not careful and perceptive enough to make it work, maybe ill try InFusion. Have you?

As to the digestion issue, I always use an excess of DNA (vector or insert) in my digestion because the efficiency of REs is so small. I design my primers and oligos so that theres like 5 bases after the RE site to make sure.

Satellite colonies have grown off the main colony after the antibiotics in the medium have been broken down during the incubation, so you get such colonies with e.g. ampicillin but not with kanamycin/chloramphenicol/tetracycline/etc.