Subcloning should be easy and fast, and work every time. The following protocols minimize the number of manipulations required to prepare DNA fragments for ligations, thereby both saving time and increasing reliability.

Preparation of DNA fragments for ligation.

1. Restriction digests:

Always cut a lot of your starting plasmids in a small volume; this will help in the gel purification of your restriction fragments by giving you a high concentration of DNA compared to agarose in your gel slice.. About 1 µg in a 20 µl reaction is good.

For double digests: you almost never have to digest with one enzyme, adjust the buffer, and digest with the second. Look in the Biolabs catalogue to find a compromise buffer, and save yourself some time.

Don't use just 1 unit of enzyme and wait an hour: your time is worth too much, and the enzymes are cheap and pure. Use about a 10-fold enzyme excess and digest about 30 min.

2. Playing with the ends.

Blunting 5' overhangs: Add 1 µl 2 mM all four dNTPs to your 20 µl restriction digest. Add 0.5-1 µl Klenow (2-5 units), and incubate at room temp for 30 min.

Blunting 3' overhangs: Add 1 µl 2 mM all four dNTPs to your 20 µl restriction digest. Add 0.5-1 µl T4 DNA polymerase, and incubate at 37℃ for 5 min. T4 polymerase has a more active 3' to 5' exo activity than Klenow, and so is preferred for this reaction, but Klenow will work.

Blunting both a 3' and 5' end in the same reaction: using either Klenow alone or T4 polymerase alone or a mixture will work okay.

Blunting an end, and then cutting with another enzyme to produce another end that is sticky: after blunting the ends produced by the first enzyme, add ~80 µl 0.3 M NaOAC pH 5.2, phenol/chloroform extract, chloroform extract, add 3 volumes EtOH, put at -20℃ for 20 min., spin 10 minutes, wash with 70% EtOH, dry, and resuspend in the appropriate buffer for the next restriction digest. Then add the second restriction enzyme and proceed. (Note: I've tried just killing the Klenow or T4 polymerase by heating to 70℃ for 20 minutes, and then adding the second restriction enzyme. This hasn't given good results, though; I think Klenow in particular is somewhat thermostable).

Phosphatasing to prevent vector reclosures: After the restriction digest, add 1 µl Biolabs calf intestine alkaline phosphatase. Incubate 37℃ for 1 hr. (In the old days I used Boehringer phosphatase: they sell a high concentration type so that you can put in ~20 units to help phosphatase blunt and 3' overhang ends, which are more resistant to phosphatasing. In the old days we also used to change to an alkaline buffer for phosphatasing, but that appears unnecessary.)

Blunting an end, and then phosphatasing: after blunting, ~kill the Klenow or T4 pol by incubating at 70℃ for 20 min, and phosphatase as usual. There is a theoretical concern here that the unused free nucleotides will compete with the DNA ends for the phosphatase, but empirically it seems not to matter. If you're worried about this you could EtOH ppt. and resuspend before phosphatasing.

Cloning Taq polymerase PCR products by T/A cloning: Taq polymerase has an efficient terminal transferase activity that adds a single nucleotide to the 3' end of blunt ended duplex DNA. This terminal transferase activity greatly prefers adding A over adding C, G, or T, so most PCR products will have single 3' A overhangs. These can be cloned efficiently into a vector containing single 3' T overhangs on its ends. You could buy kits containing prepared vectors of this structure, but you can easily produce the equivalent yourself. After cutting 1 µg of any vector with a blunt cutter (e.g. Bluescript, using EcoRV) dilute the digest with 1 volume of 1X PCR buffer, add dTTP to 0.1 mM, add 2 U Taq polymerase, and incubate at 72℃ for 20 minutes. The Taq will add a single T to the 3' ends of the vector DNA. The vector will now self-ligate very inefficiently, and should accept Taq generated PCR products. In practice, however, ligations of such vectors with PCR fragments yield only ~10-50% clones with inserts, so it is best to use blue/white selection to identify colonies with inserts; almost all the white colonies will have an insert.

3. Gel purifying DNA fragments: I generally gel purify all DNA fragments; even if I'm just linearizing a vector I run it out on a gel to purify it away from the enzymes and buffers it is in. Gel purification provides a quick way to purify the DNA, and allows you to check what your putting in to your ligation to make sure it's alright.