04/28/15
- Ryan - minipreps & digests
- Ryan - PCR & Dig/Lig (promoter insert trial #2)
Minipreps
- Use Sigma GenElute; elute with 75 μL elution solution
- BraR/MRV colonies - picked 4 additional colonies from original cloning plate
- Cut w/ E/X
- Bra/MRV 3
- Bra/MRV 4
- Bra/MRV 5
- Bra/MRV 6
- promoter/Regulator/MRV colonies
- Cut w/ KpnI/EcoRI - BbsI drop-in of promoter destroys KpnI site; empty vector will produce 2 bands, successful clones will be linearized (1 cut)
- pAub/AubR/MRV
- pBja/BjaR/MRV
- pRpa/RpaR/MRV
| Reagent
|
Volume
|
Expected:
1-4. Bra/MRV = ~3200, 776
Empty MRV E/X = ~3200
5. pAub/AubR/MRV = 4240
6. pBja/BjaR/MRV = 4099
7. pRpa/RpaR/MRV = 4116
Empty vector = ~4000, 971
|
|
| DNA(plasmid) |
3.0 μL
|
| 10X buffer |
1.5
|
| enzyme 1 |
1.0
|
| enzyme 2 |
1.0
|
| dH2O |
8.5
|
| |
15 μL --> 37°C/ ~15 min.
|
| Sample |
OD 260 |
260/280 |
ng/μL
|
| BraR/MRV #5 |
0.465 |
1.913 |
465.4
|
Conclusions
- BraR/MRV - Success! Keep colony 5 (lane 3)
- promter/Reg/MRV - none worked. Try the assembly again with PCR-amplified promoter inserts
Ryan - PCR & Dig/Lig (promoter insert trial #2)
Ryan - Receiver plasmids, assembly - REVISED STRATEGY (PCR promoters)
- Stage 1 - insert Regulator ORFS
- Phusion PCR-amplify ORFs (add 5'-E, 3'-X cut sites)
- Insert ORF(E/X) into Vector (E/X). see Vector plasmid map
- Stage 2 - insert Promoters
- PCR-amplify promoter inserts (same primers as Stage 1)
- Cut & dephos promoters with E/X
- Insert promoters(E/X) into Vector/Regulator (BbsI) via Lig/Dig cycling
Assemblies
- pAub/AubR/MRV: pAub(E/Xdp)/153 + AubR/MRV(BbsI)/4087
- pBja/BjaR/MRV: pBja(E/Xdp)/122 + BjaR/MRV(BbsI)/3977
- pBra/BraR/MRV: pBra(E/Xdp)/214 + BraR/MRV(BbsI)/3977
- pRpa/RpaR/MRV: pRpa(E/Xdp)/136 + RpaR/MRV(BbsI)/3980
- Measure conc.'s
- Values from last week, except BraR/MRV, which was measured today
| Sample |
OD260 |
260/280 |
ng/μL
|
| 1. AubR/MRV |
0.258 |
1.906 |
257.7
|
| 2. BjaR/MRV |
0.177 |
1.904 |
177.3
|
| 3. BraR/MRV #5 |
0.465 |
1.913 |
465.4
|
| 4. RpaR/MRV |
0.246 |
1.896 |
246.2
|
- pAubR, EcoRI fwd, XbaI rev
- pBjaR, EcoRI fwd, XbaI rev
- pBraR, EcoRI fwd, XbaI rev
- pRpaR, EcoRI fwd, XbaI rev
| bgcolor=#cfcfcf |
Reagent
|
bgcolor=#cfcfcf |
Vol
|
bgcolor=#cfcfcf |
Mix (x2)
|
rowspan=7 |
Expected:
1. pAubR = 153
2. pBjaR = 122
3. pBraR = 214
4. pRpaR = 136
|
| gBlock DNA (2 ng/μL) |
0.5 |
1.0
|
| 10 μM XbaI rev |
1.0 |
2.0
|
| 10 μM EcoRI fwd |
1.0 |
2.0
|
| 2x GoTaq green |
25.0 |
50.0
|
| dH2O |
22.5 |
8.7
|
| |
50.0 μL |
|
|
Thermal cycler: Labnet - GOTAQ
- 95°C 3 min.
- 30x[95°C, 30 sec; 57°C 30 sec; 72°C 30 sec]
- 72°C 3 min.
- 4°C, ∞
| Reagent
|
Volume
|
| DNA (20 ng) |
10.0 μL
|
| 10x buffer |
2.0
|
| EcoRI |
1.0
|
| XbaI |
1.0
|
| dH2O |
6.0
|
| |
20.0 μL --> 37°C/ ~15 min.
|
- Dephosphorylation (Roche)
- Assuming that shrimp alkaline phosphatase works in FastDigest buffer conditions Link
| Reagent
|
Volume
|
| DNA (digest) |
20.0 μL
|
| phosphatase |
1.0
|
| dH2O |
---
|
| |
20 μL
|
--> 37°C/ 10 min.; 75°C/ 2 min.; [final] = ~1 ng/μL
- Digestion/Ligation Reactions (borrowed from Rene's gRNA/Cas9 assembly procedure)
- Inserts are gBlocks = ~2ng/μL
- For an insert = ~200 and vector = ~4000, need 5 ng insert for 2:1 insert:vector(50 ng) ratio
- pAub/AubR/MRV: pAub(E/Xdp)/153 + AubR/MRV(BbsI)/4087
- pBja/BjaR/MRV: pBja(E/Xdp)/122 + BjaR/MRV(BbsI)/3977
- pRpa/RpaR/MRV: pRpa(E/Xdp)/136 + RpaR/MRV(BbsI)/3980
- Note: BraR cloning was not successful, omit from this round of assembly
| |
1 |
2 |
3
|
| Insert DNA |
5.0 |
5.0 |
5.0
|
| Vector DNA |
0.2 |
0.3 |
0.2
|
| 10x FD buf |
2.0 |
2.0 |
2.0
|
| 10 mM DTT |
1.0 |
1.0 |
1.0
|
| 10 mM ATP |
1.0 |
1.0 |
1.0
|
| FD BbsI/BpiI |
1.0 |
1.0 |
1.0
|
| T4 ligase (Roche) |
1.0 |
1.0 |
1.0
|
| dH2O |
8.8 |
8.7 |
8.8
|
| |
20.0 μL |
20.0 μL |
20.0 μL |
|
Thermal cycler: Labnet - BbsI Dig/Lig
- 6x[37°C, 5 min.; 23°C 5 min.]
- 4°C, ∞
|
Karmella's BioBrick Cloning
