Restriction enzymes

restriction fragments

small pieces of DNA generated when digested by restriction enzymes

EcoRV

what would the fifth restriction enzyme from E. coli strain RY13 be named?

PstI

what would the first restriction enzyme from Providencia stuartii be called?

C, T

in a recognition sequence, what can Y be?

A, G

in a recognition sequence, what can R be?

any nucleotide

in a recognition sequence, what can N be?

C, T

pyrimidines (Y)

A, G

Purines (R)

isochizomers

restriction enzymes that recognize the same sequence, but can cleave at different positions

sticky ends

DNA fragments with identical protruding termini, include 5’ and 3’ overhangs

DNA ligase

enzyme can join two different DNA fragments with compatible cohesive ends

ligation

covalent joining of annealed DNA fragments by DNA ligase

-20C

at what temperature should restriction enzymes be stored at?

directly place in ice bucket, bring with you to freezer

What should you do with a restriction enzyme when grabbing it from the -20C freezer?

yes

do you need to wear gloves when handling restriction enzymes?

digestion buffer

specific recipe of chemical requirements for restriction enzymes

1uL of 10X buffer

You want to create a 10 fold dilution of a 10X buffer with a volume of 10uL, how much buffer should you add?

50% glycerol

in solution of enzyme, increases storage stability by preventing ice crystallization at -20C

can interfere with enzyme digestions

while 50% glycerol is in the solution of the stored enzyme helping prevent ice crystallization, why should the concentration be no higher than 5% ?

½ hour

What is the standard incubation time for restriction digests for newer enzymes/what we use in class?

TE, buffer, enzyme

what is in the “premix” used when using the same RE for multiple DNA samples?

EDTA

chelates the divalent cations needed for RE activity temporarily inactivating enzyme activity

excess MgCl2

can be added to reverse inhibition of enzyme by EDTA

heat for 10-20min at 65C or 80C

While you can use EDTA to temporarily inactivate an enzyme, what can do this permenantly?

SDS, phenol

chemicals can be added to permanently inactivate enzymes, but also denatures any other enzymes present