Lab 2.2 prep

Restriction Digest

9/27 & 9/29 2022 (9/23 & 9/28 2021)

2020 experiment

Reagents & Materials

• 2 plasmids from Lab 2.1

• Wide format gel

  • 6 1% 100 mL SYBR Safe gels
  • TAE in carboy
  • 2 20-well combs per gel (buy more!)
• Restriction enzymes (get the large size!)
  • BamH1-HF
  • Kpn1-HF
  • Pvu1-HF
• Diluent B to dilute enzymes to 1/4 strength
• NEB Purple 1 Kb ladder
• Purple loading dye SDS-free (comes with enzymes)
• hot blocks for 168 reactions (!) Maybe an incubator?

Restriction Digest Volumes

Reagent	amount
Restriction Enzyme	4 µL (after dilution with Diluent B)
DNA	1 µg
10X NEBuffer (Cutsmart)	2.5 µL (1X)
Loading dye	4 uL
water to final volume	25 µL
Incubation Time	1 hour
Incubation Temperature	Enzyme dependent, generally 37C

Aliquots

16 reactions per pair (8 reactions x 2 plasmids):

• 3 single
• 3 double
• 1 triple digest
• 1 uncut

Reagent	per reaction	for 16 rxns +	for 12 pairs	for 24 pairs
Cut Smart	2.5 µL	50 µL	600 µL	1200 µL
Enzyme	4 µL (diluted)	36 µL (8 rxns)	432 µL	864 µL
Load dye	5 µL (for 25 µL rxn)	100 µL	1200 µL	2400 µL
Ladder	10 µL	25 µL	300 µL	600 µL

• For 3 single, 3 double, 1 triple digest, and 1 uncut x 2 plasmids per pair:
  • Cut smart: 50 µL (16 rxns x 2.5 µL = 40 µL)
  • Enzymes: dilute to 1/4, then give 20? µL (4 rxns x 4 µL = 16 µL)

Restriction sites

Plasmid	Bam1	Kpn1	Pvu1	plasmid length
20972 (H2B)	1724	2117	3567	6476
28009 (ecadherin GFP)	3703	3059	6007	8820

Digest fragments

Plasmid	BamH1 + Kpn1	BamH1+Pvu1	Kpn1+Pvu1	BamH1+Kpn1+Pvu1
20972 (H2B)	393, 6083	1843, 4633	1450, 5026	393, 1450, 4633
28009 (ecad)	646, 8174	2304, 6516	2948, 5872	646, 2304, 5870