Question

What are the appropriate digest conditions (buffer and temp) for BglII, HindIII, and SmaI, each individually? What conditions would you use to do a double digest with BglII AND HindIII? A double digest with HindIII AND SmaI? (Hint: Look up the enzymes and use the Double Digest Finder tool at New England Biolabs website) (2 points)

Answer 1

BglII

optimum temperature - 37° C

compatible buffer- Buffer O

it is not sensitive to heat inactivation

HindIII

optimum temperature - 37 ° C

compatible buffer - buffer R

it is sensitive to heat inactivation

Sma I

optimum temperature- 30  ° C

compatible buffer - buffer tango

it is sensitive to heat inactivation.

double digestion of BglII and HindIII

	temperature ( ° C)	buffer	time saver	heat inactivation ( ° C)
BglII	37	NE Buffer 3.1	yes	no
HindIII	37	NE Buffer 2.1	no	yes (80)

double digestion of  Hind III and Sma I

	temperature ( ° C)	buffer	time saver	heat inactivation ( ° C)
HindIII	37	NE Buffer 2.1	no	80
SmaI	25	Cut smart buffer	yes	65