2 - Molecular Biology
2 - Molecular Biology kdorfman Fri, 08/07/2020 - 15:43- H2B-mCherry (Addgene 20972) (Kan resistant)
- ecadherinGFP (Addgene 28009) (Kan resistant)
Restriction Enzymes
- PvuI-HF (cut smart)
- BamHI-HF (cut smart)
- KpnI-HF (cut smart)
A "Typical" Restriction Digest
| Reagent | amount |
|---|---|
| Restriction Enzyme | 10 units is sufficient, generally 1 µl |
| DNA | 1 µg |
| 10X NEBuffer | 5 µl (1X) |
| Total Reaction Volume | 50 µl |
| Incubation Time | 1 hour |
| Incubation Temperature | Enzyme dependent |
Gel Loading
- 1 % agarose in TAE
- 1/10,000 SYBR Safe
- 20 wells
- wide gel
- 10 uL/well
Lanes 1, 10, 20 have NEB 1Kb ladder mixed in this ratio
- 1 uL ladder
- 1 uL loading dye
- 6 uL water
| Tube | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 |
|---|---|---|---|---|---|---|---|---|
| Ingredient | uncut control | BamH1 | KPN1 | Pvu1 | BamH1 + Kpn1 | BamH1+Pvu1 | Kpn1+Pvu1 | BamH1+Kpn1+Pvu1 |
| 10X NEBuffer | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 |
| plasmid 20972 | 3 | 3 | 3 | 3 | 3 | 3 | 3 | 3 |
| BamH1 | 0 | 1 | 0 | 0 | 1 | 1 | 0 | 1 |
| KPN1 | 0 | 0 | 1 | 0 | 1 | 0 | 1 | 1 |
| PVU1 | 0 | 0 | 0 | 1 | 0 | 1 | 1 | 1 |
| water | 42 | 41 | 41 | 41 | 40 | 40 | 40 | 39 |
| Tube | 11 | 12 | 13 | 14 | 15 | 16 | 17 | 18 |
|---|---|---|---|---|---|---|---|---|
| Ingredient | uncut control | BamH1 | KPN1 | Pvu1 | BamH1 + Kpn1 | BamH1+Pvu1 | Kpn1+Pvu1 | BamH1+Kpn1+Pvu1 |
| 10X NEBuffer | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 |
| plasmid 28009 | 9 | 9 | 9 | 9 | 9 | 9 | 9 | 9 |
| BamH1 | 0 | 1 | 0 | 0 | 1 | 1 | 0 | 1 |
| KPN1 | 0 | 0 | 1 | 0 | 1 | 0 | 1 | 1 |
| PVU1 | 0 | 0 | 0 | 1 | 0 | 1 | 1 | 1 |
| water | 46 | 45 | 45 | 45 | 44 | 44 | 44 | 43 |
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 |
