I am sequentially digesting a plasmid with two incompatible restriction enzymes (NEB). The NEB protocol recommends to always carry out the first digestion with the enzyme that requires the lowest salt concentration. The first enzyme uses NEBuffer 1.1 (1X) which has 0 mM NaCl requirement whereas the second one uses NEBuffer 3.1 (1X) which has 100 mM NaCl requirement. The first digestion volume is 50 ul. The protocol recommends addition of sufficient NaCl to bring the salt concentration to that of NEBuffer 3.1 and the second enzyme thereon. If it is done so, wont the volume of the second digestion increase more than 50 ul. Wont the additional volume of 100 mM NaCl to the initial 50 ul reaction would further dilute the the buffer concentration?
http://nebcloner.neb.com/#!/protocol/re/sequential-heat/SacI,BamHI
Hi there,
You may easily prepare a 5M NaCl stock solution. Addition of 1µL of this stock to your 50µL reaction mix will adjust NaCl to 0.1M with very little effect on volume and therefore on other component concentrations.
Hi there, I have digested plasmids using NEBs buffers and enzymes before in volumes ranging from 10uL to 75uL (even though it suggests 50uL) and it has worked fine for me. You should check the efficiency on a gel afterwards though, just to be sure.
About adding extra NaCl, you should take from a sufficiently concentrated stock NaCl solution so that the additional volume doesn't alter the buffer concentration much. I would cap it at 2.5 uL. That means adding 2.5 uL of 2.1M NaCl solution. The rest of the buffer components would decrease in concentration by only 5%. Alternatively, you can add 2.5uL of a 2.11M NaCl solution and an additional 0.25uL of buffer 1.1 to compensate for the small dilution (your final volume would then be 52.75uL).
Hi there,
You may easily prepare a 5M NaCl stock solution. Addition of 1µL of this stock to your 50µL reaction mix will adjust NaCl to 0.1M with very little effect on volume and therefore on other component concentrations.
Thanks for the replies....I thought of adding 8 ul of 750 mM NaCl + 1 ul 2nd enzyme and 1 ul of first buffer to make the volume upto 60ul and also making both the buffer and NaCl concentration to 6X and 100mM respectively. So it'll be a 60 ul reaction and I suppose all the ingredients are managed.
- Badges
- Science topic
- Similar topics
- Biological Science
- Biochemistry
- Enzymology
- Enzymes