03/27/15
- Cultures - BD006/MV2 colonies (from 3/26/15 ligations), 5 mL each (4 total)
- Plasmids - DPRE minipreps (5 mL cultures)
Plasmids - DPRE minipreps
- ---Karmella 15:12, 27 March 2015 (EDT):
- Sigma GenElute Miniprep kit.
- Elute with 70μL elution buffer
- DPRE a
- DPRE b
- Digests (Fermentas FD)
- E/S to prep for insertion upstream of BD004
- Also digest BD004 w/ E/X
Reagent
|
Volume
|
Expected: 1,2. DPRE (E/S) = 1740*, 3200 3. BD004 (E/X) = 7318* *Bands cut & purified
|
30 μL/lane, 1% agarose; Ladder
|
DNA (plasmid) |
20.0 μL
|
10x buffer |
3.0
|
enzyme 1 |
1.0
|
enzyme 2 |
1.0
|
dH2O |
5.0
|
|
30 μL --> 37°C/ ~30 min.
|
Sample |
OD260 |
260/280 |
ng/μL
|
1. DPRE a mp |
0.411 |
1.89 |
411.47
|
2. DPRE b mp |
0.411 |
1.89 |
410.51
|
- Gel purification conc.'s
- Sigma gel extraction kit, eluted & back-eluted w/ 30 μL elution sln
Sample |
OD260 |
260/280 |
ng/μL
|
1. DPRE a (E/S) |
0.017 |
1.88 |
16.81
|
2. DPRE b (E/S) |
0.013 |
1.54 |
13.41
|
2. BD004 (E/X) |
0.006 |
1.85 |
6.46
|
|