Why, oh why, am I having so much trouble with ligations and transformations? - I need a little holiday help (Dec/24/2009 )

Hi All,

I am a relative newcomer and novice to molecular biology. I am working on a project using molecular methods to study bat diets: I get some bat feces, and use universal arthropod primers to amplify any insect template in the feces. Because I don't know how many different insect species DNA may be in the feces, I have been using molecular cloning -- ligating the PCR product into vectors, transforming into bacteria, and then performing colony PCR and sequencing vector with insert.

At least, this has been the *idea*. However, I'm really running into some problems with the cloning that have me flummoxed. I am using the Invitrogen TA cloning kit, with TOP10 chemically competent cells, using a protocol that a colleague developed. Specifically, although I get a *lot* of colonies (usually) by the end, they are almost always all white, and when I do colony PCR most seem to be empty vector. I would appreciate any suggestions or help. Allow me to explain what I do.

PCR Product: I'm using a "universal" invertebrate primer that amplifies a 480-bp segment of the mitochondrial COI gene. Because my template is so degraded, I tend not to get much PCR product, and in spite of my efforts to optimize, I do get dimers and the occasional off-size bat product. Because of the dimers and weak products, I have been using gel-eluted product in my ligations.

Gel elution: I usually run the product from 2 or 3 PCR reactions in one lane (because the bands are weak), in LMP agarose gel. I use a long-wavelength handheld UV illuminator to excise bands and take great pains to minimize UV exposure. (I have not yet tried the guanosine trick advocated by some here.) Use a commercial kit to clean up DNA from excised agarose.

A-Tailing: Even though I'm using a regular Taq for my PCR, I have been going ahead with A-tailing, since from what I understand, the gel elution process can cause the A overhangs to come off. (Recently, to test this idea, I did try setting up some ligations directly from the gel purified product skipping the A-tailing step, and I ended up with no colonies.) I do 20 minutes at 72 degrees, with Taq, 10X buffer, and dATP's. Before I set up the reaction, I check the DNA concentration of the eluted DNA and set up the A-tailing reaction so that I will have a 3:1 ratio of insert:vector in molar weight.

Ligation: I set up smaller volume reactions than Invitrogen suggests in their handbook, because I have limited funds and need to squeeze more reactions out of each kit; this has not been a problem for anyone else using the same protocol. Each ligation contains 1.25 ul water, 0.25 ul 10X ligation buffer, 0.5 ul PCR2.1 vector, and 0.25 ul ligase. I ligate overnight at 14 degrees.

Transformation: I use half volume (25 ul) of chemically competent cells, in 1.7 ml microcentrifuge tubes. I add 2ul of the ligation mixture, flick the tubes a couple of times gently to mix (no pipette mixing or vigorous mixing that could damage the cells) incubate on ice for 30 min, heat shock for 45 seconds, put back on ice for 90 sec, add 125 ul of SOC, then incubate at 37 degrees in a shaking incubator for 1 hour, 220 rpm. I keep the tubes on their sides.

Plating: I use regular LB/amp plates. An hour or so before, I spread 50 ul of X-gal (solution of 20 mg per ml, in dimethyl formamide) on the plates and allow it to absorb into plates; I keep the Xgal in the freezer, in small aliquots wrapped in foil, and keep the X-gal plates in the dark. Plates are kept in 37 deg incubator before plating. After the incubation I plate the entire volume of the transformation reaction (about 150 ul) on the plates and spread using glass beads, and then incubate for about 18 hours. I incubate the plates at 4C for 1-2 hours before screening colonies.

Results: I get lots (>100) of colonies; and they are nicely spread out on the plate. I can say that at least. They are all white, though. (The only exception is for the first few plates I did, before I started gel elution and then A-tailing and then would get only a few colonies -- maybe 10 at most.) I mean, not a single damn one will look even the slightest bit blue. I have used brand new Xgal in making my latest batch, and I am pretty sure that the Xgal is not the problem.

It would be OK if they where all white, and then also had my 480-bp insert. But, when I do colony PCR, most will have no insert. I am wondering if this could be a result of the A tailing? Is it possible that after the A tailing, there are too many free dATP's floating around in the soup and that somehow these are getting ligated into the vector, essentially producing empty vectors but also disrupting the gene that would let them metabolize the Xgal and turn blue?

Does anyone have any suggestions as to what to do about this?

Thanks!
Kim

Dear Kim,

as always it is essential to perform control experiments (this will help you save a lot of time, money and nerves)! The problems you describe could be due to degradation of the 3'T overhangs on the vector. This could happen when the vector is stored for too long or exposed to repeated freeze-thaw cycles. I would suggest to check the vector for self ligation (you will find the recipie in the original TA cloning manual, but its just a normal ligation reaction without insert).

Controls are the key to success

Hope this will help you get along!

Happy Xmas!

Regards,
p

Hi,

I agree with the above poster, you need to set up controls to help with troubleshooting, particularly for the transformation component. Here is what I would do when I perform a transformation:

Controls
A. vector - ligase
B. insert - ligase
C. vector + ligase
D. insert + ligase
Test Samples
A. Vector + DNA fragment (equimolar) + ligase
B. Vector + DNA fragment (three molar equivalanet of vector) + ligase
C. Vector + DNA fragment (five molar equivalent of vector) + ligase

For example:
1/ A. is the vector only control, and shows the presence of uncut DNA in the vector preparation.
2/ C. shows re-ligation of the vector, so colonies here indicate that the vector was inadequately phosphatased. When two different restriction enzymes have been used to prepare the vector, and it has not been phosphatased, colonies from this ligation show that one enzyme has digested incompetely.

Cheers,
massiveattack

I would also plate a "known blue" colony on your plates to see if your X-Gal is working. Personally, I add the X-Gal to the plates just before pouring them (like you do with the amp), and I've also never found in necessary to do tailing in TA cloning.

HomeBrew on Dec 25 2009, 08:14 PM said: