Lab 2.2 prep
Lab 2.2 prep kdorfman Wed, 08/25/2021 - 20:44Restriction Digest
9/27 & 9/29 2022 (9/23 & 9/28 2021)
Reagents & Materials
2 plasmids from Lab 2.1
-
- 6 1% 100 mL SYBR Safe gels
- TAE in carboy
- 2 20-well combs per gel (buy more!)
- Restriction enzymes (get the large size!)
- 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 |