Digest PcTF1 (BL01)
Restriction Digest Table
1. BL01 = 2500bp
| DNA(BL01) || 15.0 μL
| 10X buffer || 3.0 μL
| XbaI || 1.0 μL
| SpeI || 1.0 μL
| dH2O || 10 μL
| Total || 30 μL --> 37°C/ 15 min.
Gel Purification: BL01/V0120
- Using Prior Digest
- Placed 15μL 1kB Ladder in well one and 30μL of BL01 digest in well two.
- Ran gel at 110V for 55 minutes.
- Placed gel on UV transilluminator and cut out desired band.
0.8% agarose; cut the brightest band furthest down at ~ 2500bp; Ladder
- Used Zymo gel purification kit.
- Assumed 200mg gel, added 600μL ADB to the 1.5mL tube.
- Put on heat block at 55°C for 5 minutes, vortexed and then placed back on the heat block for an additional 5 minutes, vortexed again.
- Put ~750μL gel/ADB into spin column, centrifuged at max rpm for 30 seconds.
- Pipetted 200μL wash buffer to the tube, centrifuged at max rpm for 30 seconds. Repeated once.
- Transferred column to new labeled 1.5mL tube.
- Pipetted 15μL elution buffer to the column.
- Spun at max rpm for 30 seconds.
| Plasmid || OD260 || OD260/280 || ng/μL
| 1. Purified BL01 || 0.001 || 2.831 || 10.694
- Put marked (GelP BL01 5/15/2014) BL01 in -20°C fridge in box labeled Cameron.
- Added 10μL dH2O to two 0.5mL tubes marked 1 and 2.
- Pipetted 0.5μL BL01 into tube 1. Put both tubes on ice.
- Let DH5a-T cells thaw in ice.
- Transferred 50μL cells into each tube. Incubated for 5 minutes on ice.
- Spread sample 1 onto labeled plate 1 using sterile beads. Repeated for tube two and plate two.
- Placed in incubator for overnight growth.