03/20/15
- Plasmids - minipreps & digests
Plasmids - minipreps & digests
- Sigma GenElute Miniprep kit.
- Elute with 75μL elution buffer
- MV2 a
- MV2 b
- BD004 a
- BD004 b
- BD006 a
- BD006 b
Reagent
|
Volume
|
Expected: 1,2. MV2 (E/X) = 4500 3,4. BD004 (E/S) = 4116, 3200 5,6. BD006 (E/S) = 4182, 3200
|
30 μL/lane, 1% agarose; Ladder
|
DNA (plasmid) |
20.0 μL
|
10x buffer |
3.0
|
enzyme 1 |
1.0
|
enzyme 2 |
1.0
|
dH2O |
---
|
|
30 μL --> 37°C/ ~30 min.
|
- Conclusions
- MV2 is correct
- BD004 - most likely missing the D11.12 fragment. Sequence results from Meli'sa's past work agrees with the short fragment size on the gel.
- BD006 is correct
- Miniprep conc.'s
- Combined the pairs of preps. Pairs look identical.
Sample |
OD260 |
260/280 |
ng/μL
|
1. MV2 mp |
0.132 |
1.85 |
132.262
|
2. BD004 mp |
0.144 |
1.91 |
143.967
|
3. BD006 mp |
0.078 |
1.83 |
78.413
|
Assemblies
- BD006/MV2: BD006(E/S)/4000 + MV2(E/X)/4500
- Gel purification: Sigma Gel extraction kit, elute and back-elute with 40 μL elution buffer
Sample |
OD260 |
260/280 |
ng/μL
|
1. MV2-band1 (E/X) |
0.033 |
0.58 |
32.94
|
2. MV2-band2 (E/X) |
-0.01(?) |
--- |
---
|
3. BD006-band1 (E/S) |
-0.005(?) |
--- |
---
|
4. BD006-band2 (E/S) |
-0.01(?) |
--- |
---
|
|