While T-vector cloning is commonly used for PCR-amplified inserts, restriction enzymes still have their uses. For example, you can ensure directional cloning if you digest a vector with the same two enzymes like BamHI and EcoRI that are used to digest your insert. This way, the insert can only be cloned in one direction. However, there are other cloning techniques that can be used to modify the end of vectors and inserts after restriction enzyme digestion and prior to ligation.
Dephosphorylation of 5´ Ends
If the ends of the prepared cloning vector are identical (e.g., cut with a single restriction enzyme), treat the vector with a phosphatase like TSAP Thermosensitive Alkaline Phosphatase (Cat.# M9910) to remove the phosphate groups from the 5′ ends and prevent self-ligation of the vector. For linear vectors with unique 5′ ends, dephosphorylation is not necessary. The dephosphorylation reaction can be performed directly in restriction enzyme buffer so the vector can be cut and dephosphorylated at the same time.
- As a general guideline, for reactions containing up to 1µg of DNA, add 15 units of restriction enzyme and 1–2µl of TSAP (depending on the reaction buffer used) to the vector DNA in a total reaction volume of 20–50µl. Set up the reaction in the appropriate 1X Promega restriction enzyme reaction buffer.
- Incubate the reaction at 37°C for 15 minutes. This is sufficient to digest and dephosphorylate all vector DNA overhang types (3´, 5´ or blunt).
- Heat-inactivate the TSAP and restriction enzyme by incubating the reaction at 74°C for 15 minutes. For restriction enzymes that cannot be heat-inactivated, clean up the digest using a fragment purification kit like the Wizard® SV Gel and PCR Clean-Up System (Cat.# A9281).
Converting a 5´ Overhang to a Blunt End
Depending on cloning strategy, there may be times when a restriction enzyme leaves an overhang but the cloning vector is blunt ended. Both Klenow (DNA Polymerase I Large Fragment) and T4 DNA Polymerase can be used to fill 5′-protruding ends with deoxynucleotide triphosphates (dNTPs). Below is one method to modify the ends of an insert prior to ligation into a blunt-end vector.
- Digest the insert of interest with restriction enzyme that leaves a 5′ overhang.
- Initiate the fill-in reaction directly in the restriction enzyme buffer supplemented by adding 40µM of each dNTP and 1 unit of Klenow Fragment per microgram of DNA.
- Incubate the reaction at room temperature for 10 minutes.
- Stop the reaction by heating at 75°C for 10 minutes.
Converting a 3´ Overhang to a Blunt End
For those enzymes that leave a 3′ overhang, T4 DNA Polymerase has a 3’→5′ exonuclease activity that will, in the presence of excess dNTPs, convert a 3′-protruding end to a blunt end.
- Digest DNA with restriction enzyme that generates 3′-protruding ends.
Leave the DNA in restriction enzyme buffer, exchange the buffer for 1X T4 DNA Polymerase Buffer or gel purify the desired fragment.
- Add 5 units of T4 DNA Polymerase per microgram of DNA and 100µM of each dNTP.
- Incubate at 37°C for 5 minutes.
Note: At high concentrations of dNTPs (100µM), degradation of the DNA will stop at duplex DNA; however, if the dNTP supply is exhausted, the very active exonuclease activity (200 times more active than that of DNA polymerase I) will degrade the double-stranded DNA.
- Stop the reaction by heating at 75°C for 10 minutes or adding 2µl of 0.5M EDTA.
For more information on these methods and the topic of cloning, read the Cloning chapter of our Protocols and Applications Guide.
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