05/15/15
- Ryan - assembly (traditional) for Receiver plasmids
Ben - assembly for luc replacement
- Cas-tone - minipreps & digests
Assemblies
- pAub-AubR_MRV: pAub-PCR(E/S dp)/153 + AubR/MRV(BbsI)/4087
- pBja-BjaR_MRV: pBja-PCR(E/S dp) + BjaR/MRV(BbsI)/3977
- pBra-BraR_MRV: pBra-PCR(E/S dp) + BraR/MRV(BbsI)/3977
- pRpa-RpaR_MRV: pRpa-PCR(E/S dp) + RpaR/MRV(BbsI)/3980
- DBN007_pSB1A3: DBN006(BsaI)/1972 + DBN001(BsaI)/1267/1069(2336)
- AubR/MRV(BbsI)/4087
- BjaR/MRV(BbsI)/3977
- BraR/MRV(BbsI)/3977 (two bands, isolate both)
- RpaR/MRV(BbsI)/3980
DBN006(BsaI)/1972 (this is in V0120...need PCR fragment)
- DBN001(BsaI)/1267/1069(2336) (two bands, isolate both)
Reagent
|
Volume
|
30 μL/lane, 1% agarose; Ladder -->
|
DNA (plasmid) |
15.0
|
10x buffer (FD) |
3.0
|
enzyme 1 |
1.0
|
dH2O |
11.0
|
|
30 μL
|
--> 37°C/ ~30 min.
Sample |
OD260 |
260/280 |
ng/μL
|
1. AubR/MRV(BbsI) |
0.051 |
1.924 |
51.4
|
2. BjaR/MRV(BbsI) |
0.038 |
1.983 |
38.0
|
3. BraR/MRV(BbsI) |
0.134 |
1.882 |
133.7
|
4. RpaR/MRV(BbsI) |
0.047 |
1.858 |
46.7
|
5. DBN001 (BsaI) |
0.017 |
2.176 |
16.8
|
- Ligations ("traditional")
- pAub-PCR(E/S dp)/153 + AubR/MRV(BbsI)/4087
- AubR/MRV(BbsI)/4087
- pBja-PCR(E/S dp) + BjaR/MRV(BbsI)/3977
- BjaR/MRV(BbsI)/3977
- pBra-PCR(E/S dp) + BraR/MRV(BbsI)/3977
- BraR/MRV(BbsI)/3977
- pRpa-PCR(E/S dp) + RpaR/MRV(BbsI)/3980
- RpaR/MRV(BbsI)/3980
Reagent |
Rxn1 |
Rxn2 |
Rxn3 |
Rxn4 |
Rxn5 |
Rxn6 |
Rxn7 |
Rxn8
|
Insert DNA |
2.0 |
--- |
2.0 |
--- |
2.0 |
--- |
2.0 |
---
|
Vector DNA |
1.0 |
1.0 |
1.0 |
1.0 |
0.5 |
0.5 |
1.0 |
1.0
|
2x lgn buf (Roche) |
5.0 |
5.0 |
5.0 |
5.0 |
5.0 |
5.0 |
5.0 |
5.0
|
T4 ligase (NEB) |
1.0 |
1.0 |
1.0 |
1.0 |
1.0 |
1.0 |
1.0 |
1.0
|
dH2O |
1.0 |
1.0 |
1.0 |
1.0 |
1.5 |
1.5 |
1.0 |
1.0
|
|
10.0 μL |
10.0 μL |
10.0 μL |
10.0 μL |
10.0 μL |
10.0 μL |
10.0 μL |
10.0 μL
|
- Add 50 μL DH5α-turbo; ice 5 min.
- Plate on 100 μg/mL amp
- Grow overnight @37°C
RESULTS
- Success! Ligation plates = ~50 colonies, Neg ctrls = 0 - 3 colonies
Cas-tone - minipreps, digests
- Minipreps
- Sigma GenElute mini kit; elute w/ 75μL elution sln.
- Check with E/S digests - SpeI site (@250) is destroyed and new SpeI site (@844) is gained in successful ligation
- CMV-dCas9-VP64 1-1
- CMV-dCas9-VP64 1-2
- CMV-dCas9-VP64 2-1
- CMV-dCas9-VP64 2-2
Reagent
|
Volume
|
Expected: 1-4. CMV-dCas9-VP64 = ~5400, 3016, 1363 dCas9-VP64 = 4697, 3016, 2099
|
15 μL/lane; 1% agarose; Ladder
|
DNA(plasmid) |
2.0 μL
|
10X buffer |
1.5
|
EcoRI |
1.0
|
SpeI |
1.0
|
dH2O |
9.5
|
|
15 μL
|
--> 37°C/ ~10 min.
CONCLUSIONS
- Success! Lane 4. Keep clone 2-2
|