05/06/15
- Ryan - minipreps & digests for promoter/Regulator/MRV clones
Minipreps
- Use Sigma GenElute; elute with 75 μL elution solution
- Cut w/ E/KpnI
- See my version of MRV map: https://benchling.com/s/oJ73uatK/edit
- Note: BbsI drop-in of promoter knocks out KpnI site (54); no E, X, or S sites are restored by promoter insert
- Note: EcoRI (1025) and XbaI (1040) were maintained after Regulator insertion
- pAub-AubR/MRV
- pBja-BjaR/MRV
- pBra-BraR/MRV
- pRpa-RpaR/MRV
| Reagent
|
Volume
|
Expected:
1,2. pAub/AubR/MRV = 4240
3,4. pBja/BjaR/MRV = 4099
5,6. pBra/BraR/MRV = 4191
7,8. pRpa/RpaR/MRV = 4116
9. Empty MRV = 2230, 971
np pro. Reg/MRV = ~4000, 971
|
15 μL/lane; 1% agarose; Ladder
|
| DNA(plasmid) |
3.0 μL
|
| 10X buffer |
1.5
|
| EcoRI |
1.0
|
| KpnI |
1.0
|
| dH2O |
8.5
|
| |
15 μL
|
--> 37°C/ ~10 min.
| Sample |
OD 260 |
260/280 |
ng/μL
|
| 1. pAub-AubR/MRV-1 |
0.152 |
1.930 |
152.3
|
| 2. pAub-AubR/MRV-2 |
0.079 |
1.992 |
78.6
|
| 3. pBja-BjaR/MRV-1 |
0.093 |
1.989 |
92.7
|
| 4. pBja-BjaR/MRV-2 |
0.043 |
1.934 |
43.1
|
| 5. pBra-BraR/MRV-1 |
0.070 |
2.021 |
69.9
|
| 6. pBra-BraR/MRV-2 |
0.030 |
1.946 |
29.8
|
| 7. pRpa-RpaR/MRV-1 |
0.093 |
1.956 |
92.6
|
| 8. pRpa-RpaR/MRV-2 |
0.033 |
2.000 |
32.7
|
Conclusions
- Yields low b/c cultures were only grown for ~7 hours
- Lane 1: looks like AubR/MRV (modular receiver vector) without a promoter insert
- Lanes 2 - 8: very confusing, cannot interpret this with current information
- Lane 9: appears as expected for empty MRV
- Re-do dig/lig, include negative controls for each unique vector
|
Karmella's BioBrick Cloning
Main project page
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