Adding BSA to a reaction lessens enzyme loss on tube and pipette tip surfaces. BSA stabilizes enzymes in reaction. The stabilizing effects are most pronounced in overnight reactions (Robinson D.
How much BSA do you add to restriction digest?
3 µL 10x BSA (if recommended)
What is needed for restriction digestion?
The components of a typical restriction digestion reaction include the DNA template, the restriction enzyme of choice, a buffer and sometimes BSA protein. The reaction is incubated at a specific temperature required for optimal activity of the restriction enzyme and terminated by heat.
What enzyme is used in restriction digest?
restriction endonuclease restriction enzyme, also called restriction endonuclease, a protein produced by bacteria that cleaves DNA at specific sites along the molecule.
What are the ingredients of the buffer used in restriction digestion?
Restriction enzyme digestion buffer
- 10 mM Tris-Cl (pH 7.4)
- 0.2 mM EDTA.
- 0.2 mM EGTA.
- 0.15 mM spermine.
- 0.5 mM spermidine.
- 1 mM β-mercaptoethanol.
Why do we digest PCR products?
For cloning applications, purification of PCR products prior to digestion is necessary to remove the active thermophilic DNA polymerase present in the PCR mixture. DNA polymerases may alter the ends of the cleaved DNA and reduce the yield of ligation.
How can we prevent restriction digestion?
Protocol for DNA Digestion with a Single Restriction Enzyme Incubate the reaction at digestion temperature (usually 37 °C) for 1 hour. Stop the digestion by heat inactivation (65 °C for 15 minutes) or addition of 10 mM final concentration EDTA. The digested DNA is ready for use in research applications.
Does PCR use restriction enzymes?
Restriction enzymes can also be used to generate compatible ends on PCR products. In all cases, one or more restriction enzymes are used to digest the DNA resulting in either non-directional or directional insertion into the compatible plasmid.