Summary
- Miniprep on ligated plasmid CMV MV9 liquid culture.
- Gel purification from prior CMV miniprep.
Performed mini-prep on each of the four picked colonies.
- Ran concentration check on each:
Plasmid |
OD260 |
OD260/280 |
ng/μL
|
1. Colony 1 |
0.005 |
1.211 |
4.895
|
2. Colony 2 |
0.003 |
1.273 |
2.963
|
3. Colony 3 |
0.034 |
1.888 |
33.572
|
4. Colony 4 |
0.007 |
-5.308 |
7.215
|
Digest CMV
Restriction Digest Table
- Checked plasmid minipreps with EcoRI/PstI digests
Reagent
|
Volume
|
Expected: 1. CMV = 588bp
|
DNA(CMV) |
25.0 μL
|
10X buffer |
3.0 μL
|
EcoRI |
1.0 μL
|
PstI |
1.0 μL
|
dH2O |
0 μL
|
|
15 μL --> 37°C/ 15 min.
|
Gel Purification: CMV/V0120
- Using Prior Miniprep Digests
- CMV culture 1 - check with E/P
- Used previously digested CMV.
- Placed 15μL 1kB Ladder in well one and 30μL of CMV digest in well two.
- Ran gel at 110V for 30 minutes.
- Placed gel on UV transilluminator and cut out desired bands.
15 μL/lane; 1% agarose; cut the brightest band furthest down at ~ 588bp; Ladder
|
- Used Zymo gel purification kit.
- Assumed 200mg gel, added 600μL ADB to each 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 each tube, centrifuged at max rpm for 30 seconds. Repeated once.
- Transferred columns to new labeled 1.5mL tube.
- Pipetted 15μL elution buffer to each column.
- Spun at max rpm for 30 seconds.
- Put marked (CMV gel pur2 3/22) CMV in -20°C fridge in box labeled Cameron.
|