This month's questions from the Molecular Biology Forums (online at molecularbiology.forums.biotechniques.com) come from the “General Methods” section. Entries have been edited for concision and clarity. Mentions of specific products and manufacturers have been retained from the original posts, but do not represent endorsements by, or the opinions of, BioTechniques.

Molecular Biology Techniques Q&A

What can cause a blunt-end ligation to fail? (Thread 22477)

Q I am trying to ligate an insert into a vector with blunted ends. Following ligation, I didn't get any colonies on my selection plates. How can I improve the efficiency of my ligation? Is the end-filling step or the ligation step more likely to cause the problem?

A It might actually be caused by your competent cells. If the problem was with the fill-in step, you would get many colonies containing the parental vector. If you want to check to see if the ligation worked, just run the product on a gel and look for bands of the correct size.

A You should also double check that you are using the correct antibiotics and that they are fresh.

Q I set up a control to test the competency of my cells and got several colonies, but still no colonies following ligation. I also tested the antibiotics and it seems they are fine and correct for the vector I'm using.

A In my experience, buffer conditions (salts and pH) are critical for successful ligations. For example, if more than 10% of your ligation volume is DNA from a stock stored in a buffer like TE or EB, your ligation may be severely inhibited. For this reason, I elute exclusively with pure, sterile water. You should never use non-purified restriction products in a ligation under any circumstances.

A You could try adding a molecular excluder like polyethylene glycol (PEG) into the ligation reaction. Or you could try increasing the enzyme concentration.

A Was your vector cut with an enzyme that gives a 5′ overhang? What was the enzyme and how did you fill it in? If the end-filler does not phosphorylate the strand, the fragment will not be capable of ligation. Also, you should be certain of the enzyme activity because enzymes that fill 5′ overhangs will not produce blunt ends if you have a 3′ overhang.

A DNA polymerase can fill in sticky ends, but it does not create a phosphorylation. If you have a vector fragment cleaved by a restriction enzyme, phosphorylation shouldn't be an issue. T4 DNA polymerase will take care of both 3′ and 5′ overhangs.

How can I increase the probability of incorporating my insert in blunt-end ligation? (Thread 21702)

Q I want to subclone three PCR products from 5′ and 3′ RACE. After gel purification, I set up the blunt-end ligation following the manual that came with the CloneJET PCR cloning kit from Fermentas. After heat shock transformation, I only got about 30 colonies on each plate. I picked all of them for all three ligations and screened by PCR. Only three colonies contained the vector control from the kit. None of the clones contained the insert.

I tried again with new competent cells and the vector controls worked, but none of the colonies carried the insert. Could someone who is familiar with the kit offer some suggestions?

A Did you treat your PCR fragments with T4 polynucleotide kinase? Are your primers phosphorylated?

Q In accordance with the manual, we treated the PCR product with “DNA-blunting enzyme.” My primers are not phosphorylated.

A Your vector must have been dephosphorylated so your PCR fragments need to be phosphorylated before they can be ligated to the vector. Since your primers are not phosphorylated, you will need to treat the PCR product with T4 polynucleotide kinase. Denature the enzyme with heat or gel purify your DNA and then ligate it into the vector.

A The vector in the kit you are using selects for inserts only if it has been properly cut, dephosphorylated, and gel-purified. PCR screening will show if the insert is the correct size, but you should sequence a few of the clones to see what's happening at the insertion site.

A PCR screening of colonies and DNA sequencing of the purified plasmid both require annealing of a primer that is based on the vector sequence flanking the insertion site. If the PCR screening is negative, you will not get any sequence representing the insert.

Q I understand, but the online protocol for the product does not suggest that the vector is dephosphorylated.

A The vector uses a lethal gene to suppress the growth of colonies resulting from self-ligation. But you still need to use excess insert to decrease the probability of self-ligation, which is an inefficient way to clone. I recommend dephosphorylating the vector.

A If your positive control works, why don't you try running the positive control DNA on an agarose gel alongside your sample? That should show if your insert is indeed added at the right concentration. Sometimes the solutions to ligation problems can be counterintuitive: you think you need more insert when actually you need a lot less. Are you making your own competent cells?

Q I checked the amount of insert and I am using almost 5× more than the vector. I think it should be enough. I wrote to Fermentas to ask for advice, and the company recommended that I use another type of competent cells. I tried that and did get more colonies, but still no inserts.

A If cost is an issue, 50 µL One Shot competent cells can be split into two tubes of 25 µL each and used for two ligation products (1 µL each) without any other changes in the protocol. You should do the two transformations at the same time because refreezing the unused competent cells will result in a loss of efficiency.

A In blunt-end ligation, nonspecific nucleases can chew off a few bases, allowing the vector to self-ligate. If that happens, the lethal gene will not eliminate parental vectors because it is out of frame.