CIAP is unnecessary, if you ask me. Cloning into single-digested vectors is (usually) not that difficult.

- Include 1 ng of an uncut plasmid control during transformation and use it to estimate transformation frequency. Your cells might have gone bad, or there might be something wrong with the procedure.

- If transformation frequency is OK, then include +/- ligase reactions using only your vector. Your digested/religated vector (No CIAP) should yield 50- to 100-fold more colonies than the digested vector without ligase. If not, there is something wrong at the ligation step and you should start troubleshooting from there (Bad ligase, or bad ATP, or the previous digestion was poor, or there is nuclease contamination at some step).

- If your cells are fine and the ligations work but still no dice, probably your insert is the problem. Perhaps too much UV irradiation if gel-purifying? Also check complementarity of the ends of the fragment to those in the vector (BstEII recognizes a degenerate site, so it does not follow that they are complementary, even if cut with the same enzyme). Also check that you've left enough space from the recognition sequence to the end of the undigested fragment.

Hope it helps.

Totally, agree with Alejandro Martin. My two cents: always designing your primer with additional bases (3-5) after the restriction site would be helpful for the digestion...And you can try ligation independent cloning if you are frustrated with the ligation.

The advice of the previous researchers is good. When I have had difficulty cloning a digested PCR product, I have found that it really helps to clone the PCR product into a TA vector designed for PCR products and then digest from the TA vector and ligate into the desired vector. If you use a TA cloning vector, you may have to add As to your PCR product first, depending on the polymerase you used for the initial amplification.

Thank you all for your great tips!, I added 6 extra bp to my primers for the restriction enzyme site so I think is ok (unless the primers are not well synthesised) My next step was precisely to try with new competent cells as I check with a positive control (uncut plasmid) and got no colonies as well, so im afraid my cells have gone bad, I'm using TopTen E. coli cells as they said they are good for big plasmids (mine is 10 kb), if this doesnt work then I'm gonna try using TA cloning, I'm using Pfx platinum polymerase as I need high fidelity for cloning so I'm gonna have to add 3'A to do this.

Shu Xu, can you explain to me what is this ligation indepent cloning?, I think I have an idea of what you mean but I wanna know for sure =P.

Thx all!

here is a good link to star with.

http://bitesizebio.com/articles/ligation-independent-cloning-primer-design/

Good luck!

We are using lately CloneJet PCR cloning kit from Thermo Scientific (http://www.thermoscientificbio.com/molecular-cloning/clonejet-pcr-cloning-kit/) which is great and fast for cloning of PCR products generated with any thermostable DNA polymerase. The kit is not coming with competent frozen E.coli cells, so we use our own Top10 chemically induced cells with good efficiencies. For Sequencing, pJet sequencing primers are required. We are obtaininig great results ans the kit is much cheaper than other PCR cloning kits in the market

Why do you want to clone with such an enzyme? The restriction site of BstEII seems to be outside the MCS. Have you used the proper antibiotic (Kan)?

Hello Anca, yes in my strategy I have to use BstEII as I want to introduce a gen which has to be in frame with the CaMV35S promoter (fused with the GFP), and I'm selecting with Kanamycine 50 ug/mL.

THX!

Hi Alejandro. Here can be your problem. If you only added the recognition BstEII target to the beginning of the primer, the enzyme could be unable to cut being so close to the end. NEB recomends adding extra 6 bases before the recognition target (https://www.neb.com/tools-and-resources/usage-guidelines/cleavage-close-to-the-end-of-dna-fragments). Good luck.

Have you done the restriction at optimal temperature (e.g. 60oC for NEB enzyme) ? Checked the linearization on the gel, and then purify the plasmid from the gel. Ligate the insert in the vector at the molar ration 10 insert:1 vector. Use fresh ligase buffer and do not melt the ligation buffer in the hand, but on ice. I am using Fast ligation kits from Promega or Fermentas, and they are very nice (fast and efficient). Is your insert in frame if you use PmlI? Good luck!

Hello!

>Gonzalo: thank you for the advice!, I added 6 bp at each primer right before the recognition sequence for BstEII as recommended =P, so i hope this is not the problem =)

>Anca: I'm using BstEII (Eco91l) from Thermoscientific as I cant afford NEB enzymes unfortunately (very expensive in my country), their protocol suggest digestion at 37 ºC as this is an Isoschizomer.

I checked on an agarose gel for a linearized vector and I verified it. Yesterday I tried a new ligation protocol and I saw a fragment with a size which is equal to my vector + insert so I hope I will get some colonies =D, I'm using a 3 to 1 insert - vector ratio but I'll try as you suggest 10 to 1, I'm also using fast ligation kit from promega, it is indeed quite handy. I'll tell you if I'm succesful!

Thx!

About the PmII, it wuold be in frame but It will remove part of the sequence for the GFP protein and I want to use it as a visual marker =D.

Out of curiosity: What is different in your new ligation protocol?

Hello Christian, I tried ligating using the fast ligation kit (promega) and i did it overnight at 4 ºC, I was doing it with the normal T4 DNA ligase and I was doing it for a few hours before trying the transformation and coudlnt see any bands in the gel of the expected size and no colonies.

In our hands, LigaFast kit from Promega works very fine at room temperature for 15-20 min, even for blunt ligation. I think this is the best ligase. The problem is that the buffer is 2x, and you have to have the insert with a good concentration.

The only thing that I can think of is BstEII is not cleaving your PCR product for some reason.

So i finally manage to get positive clones, after some experiments I found out that the working insert:vector ratio for me was 3:1, and for some reason I was only able to obtain positive clones in DH5a cells but none in Top10, I decided to switch cells after everything else failed and it worked right away. My insert was a 1114 bp pcr product and my vector has 10549 bp which is quite big =D.

now I would like to ask about concatemers, having this structure in my insert can be a problem?, even if I'm only using this as an intermediate step for a substitution cloning strategy?, I'm asking because when i digested my new construct I found that there is an extra band which fits with the presence of a concatemer and got me worried.

Thank you!

I forgot to mention that I also changed the amount of total DNA i was working with, so I worked with 65 ng total DNA, 15 ng of my digested insert and 50 ng of my digested vector in a 10 uL reaction for the ligation and it worked.