How do you inactivate BamHI?

Only small amounts of BamHI (up to 10 units) can be inactivated at 80°C in 20 min. To prepare the digested DNA for electrophoresis: – stop the digestion reaction by adding 0.5 M EDTA, pH 8.0 (#R1021), to achieve a 20 mM final concentration.

What is the buffer for restriction enzyme digestion?

Buffers containing low concentrations of EDTA (1mM) are often used to protect DNA from nuclease degradation during storage, but EDTA can interfere with restriction enzyme digestion if the final concentration in the reaction is too high.

Is BamHI affected by methylation?

BamHI cannot methylate this modified sequence. Methyltransferase/endonuclease pairs which differ in their sensitivity to non-canonical methylation can be exploited to generate rare DNA cleavage sites.

What is BamHI restriction enzyme?

BamHI (from Bacillus amyloliquefaciens) is a type II restriction endonuclease, having the capacity for recognizing short sequences (6 bp) of DNA and specifically cleaving them at a target site. This allows the DNA to maintain its normal B-DNA conformation without distorting to facilitate enzyme binding.

Can BamHI be heat inactivated?

Description. Thermo Scientific BamHI restriction enzyme recognizes G^GATCC sites and cuts best at 37°C in its own unique buffer. See Reaction Conditions for Restriction Enzymes for a table of enzyme activity, conditions for double digestion, and heat inactivation for this and other restriction enzymes.

Does BamHI have star activity?

The minimum number of units of BamHI required to produce star activity in the recommended NEBuffer r3. 1 is (32 units). The minimum number of units of BamHI required to produce star activity in rCutSmart Buffer is (4 units).

What does Neb buffer do?

For greater flexibility, NEB provides a selection of buffers for optimal enzyme activity, as well as for use with its protein expression and purification, cloning and RNA products. These buffers are available separately, or in bulk volumes, upon request.

What sequence does BamHI cut?

BamHI binds at the recognition sequence 5′-GGATCC-3′ , and cleaves these sequences just after the 5′-guanine on each strand. This cleavage results in “sticky ends” which are 4 b.p. long.

Can restriction enzymes cut methylated DNA?

To cleave with a restriction enzyme which is sensitive to the Dam or Dcm methylation, DNA should be purified from dam-, dcm- E….Digestion of Methylated DNA.

Can EcoRI be heat inactivated?

Description. Thermo Scientific EcoRI restriction enzyme recognizes G^AATTC sites and cuts best at 37°C in its own unique buffer. See Reaction Conditions for Restriction Enzymes for a table of enzyme activity, conditions for double digestion, and heat inactivation for this and other restriction enzymes.