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

Screening colonies