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TA cloning

TA cloning protocol

This protocol uses Promega's pGEM-T kit (#A3600).

PCR

For TA cloning, it is optimal if the PCR primers have G's at the 5' end as this will maximize the probability of Taq polymerase adding the terminal A overhang. If you are using a proofreading polymerase, add a bit of normal Taq to the mixture as Taq adds the terminal A's. Dynazyme EXT is a mixture of enzymes which already includes Taq capable of adding the A overhangs. To facilitate the A addition, add a 10 minute extension at 72C to the end of the PCR cycling. Usually, 50 ul PCR reactions provide sufficient product to clone.

Check the PCR reaction by running 5 ul out on Seakem gel to see if product is a single band.

There are several choices to clean up the product. The first is to use a Millipore Ultrafree spin column to wash out the primers. If the product is clean, this will work fine. If there are smaller fragments in the PCR, they will clone preferentially and decrease the probability of getting the larger fragments cloned. Another option is to run 45 ul out on a Seaplaque gel. Cut out the band and extract the DNA with a Qiaquick gel clean up spin column. You can also use Seakem (but not Seakem LE) if you have high quality grade Seakem agarose.

Quantify the DNA with a spectrafluorometer.

It is also possible to TA clone DNA fragments not generated by PCR (such as sheared DNA). To TA clone, the DNA should be incubated with Taq and ATP for 30 min to add the A's.

Ligation

For the ligation reaction, the optimal molar ratio of PCR / vector is 3:1. The pGEM-T vector is 3 kb and the kit suggests adding 50 ng (1 ul) of the vector. To get 3:1 molar ratios, you need 50 ng for a 1kb fragment and 25 ng for a 500 bp fragment. Do not add too much PCR insert as this leads to clones which do not PCR amplify or sequence (they act like clones which are double in size on an agarose gel). I have never had a problem adding less than 100 ng of insert.

Set up a 10 ul ligation reaction. This can be done using either 2X ligation buffer or 10X ligation buffer. The 2X works fast and is optimized for short ligation times at room temp, however, the 10X buffer works fine, especially overnight. Both work for overnight ligations at 4 C. If you need to put in more volume of PCR than possible with the 2X buffer, switch to 10X buffer. You can also compensate by setting up larger volume ligations and adjusting the buffer concentration accordingly.

Note: If you see precipitate in the ligation buffer, warm it briefly at 37C to dissolve the salts.

Sample ligation reactions:

 Reagent  High concentration PCR Low concentration PCR
 pGEM-T (50 ng) 1 ul 1 ul
PCR product 2 ul (= 100 ng) 8 ul (= 100 ng)
 ligation buffer 5 ul (2X) 1.2 ul (10 X)
T4 DNA ligase 1 ul 1 ul
 H2O  1 ul  0.8 ul
Total 10 ul  12 ul

Transformation

The pGEM-T vector is compatible with JM109 chemically competent cells which you can buy with the kit from Promega. Alternatively, you can also make electrocompetent DH10B cells (I think also DH5a) which is much cheaper and higher efficiency than the JM109's.

Make agar plates several days ahead. The day of transformation, put plates in 37C to dry for an hour or two. Around 1 p.m., prepare the plates with IPTG and Xgal mix. Add 40 ul of 20 mg/ml Xgal + 8 ul of 100 mg/ml IPTG. Spread with sterile spreader. Put back at 37 C till needed.

Around 2 p.m. start transformations. Thaw cells on ice. Add 1 ul of ligation reaction. You can also dilute the ligation reaction by 2-4 fold and then use 1 ul of that mixture. Chemically or electrically transform cells. Add 1 ml SOC. Transfer to snap cap tube and shake for 1 hr. The kit says to grow for 1.5 hrs, but this only seems to lead to duplicate clone copies when making libraries etc. One hour is better.

Plate 50-100 ul on prepared plates and leave at 37C overnight.

Screening colonies

Colonies containing insert will be white and not blue (or only faintly blue). Put plates in refridgerator for few hours to bring out the blue coloration. Colonies can be picked into 50-80 ul of LB/amp (may want to include 7.5% glycerol) in 96 well round bottom plates and grown for 2-3 hours. PCR test for insert using plasmid primers (such as M13R and T7) and 2 ul of the LB culture mixture in a 25 ul reaction. With the colonies grown up in 96 well plates, this is easily done with a multi-channel pipettor.


3/02 KC