Lab 2.2 prep

Lab 2.2 prep kdorfman Wed, 08/25/2021 - 20:44

Restriction Digest

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

2020 experiment

Reagents & Materials

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