04/21/15
- Ryan - regulator assemblies
- Rene - check gRNA assemblies; digests & sequencing
- LCR Development - Phusion PCR
Ryan - Receiver plasmids, assembly
- Stage 1 - insert Regulator ORFS
- DONE - 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
- Cut & dephos promoters with E/X
- Insert promoters(E/X) into Vector/Regulator (BbsI) via Lig/Dig cycling
- Clean up PCR products
- Zymo clean and concentrator
- Elute w/ 25 μL dH2O
- AubR/MRV: AubR-PCR(E/X)/851 + MRV(E/X)/3201
- BjaR/MRV: BjaR-PCR(E/X)/776 + MRV(E/X)/3201
- BraR/MRV: BraR-PCR(E/X)/776 + MRV(E/X)/3201
- RpaR/MRV: RpaR-PCR(E/X)/779 + MRV(E/X)/3201
- AubR, 851 bp
- BjaR, 776 bp
- BraR, 776 bp
- RpaR, 779 bp
- Modular Receiver Vector (MRV)
Reagent
|
Rxn 1-4
|
Rxn 5
|
DNA |
10.0 |
5.0
|
10X buffer |
3.0 |
3.0
|
EcoRI |
1.0 |
1.0
|
XbaI |
1.0 |
1.0
|
dH2O |
15.0 |
20.0
|
|
30.0 |
30.0
|
- Column clean-up
- Zymo clean and concentrator
Sample |
OD260 |
260/280 |
ng/μL
|
1. AubR-PCR (E/X) |
-0.005 |
5.875 |
-5.001
|
2. BjaR-PCR (E/X) |
-0.005 |
9 |
-4.788
|
3. BraR-PCR (E/X) |
-0.005 |
7.143 |
-5.291
|
4. RpaR-PCR (E/X) |
-0.003 |
29 |
-3.052
|
5. MRV (E/X) |
0.007 |
1.388 |
7.11
|
- Note: unsure why concentrations so low. Proceed with ligations anyway, see what happens.
|
Rxn 1-4 |
Neg
|
Insert DNA |
3.0 |
---
|
Vector DNA (25 ng) |
3.5 |
3.5
|
2x lgn buf (Roche) |
7.5 |
7.5
|
T4 ligase (NEB) |
1.0 |
1.0
|
dH2O |
--- |
3.0
|
|
15 μL |
15 μL
|
- Transform 50 uL DH5α-turbo
- Plate on 100 μg/mL amp
RESULTS (4/22/15)
- AubR/MRV: 11 colonies
- BjaR/MRV: 11 colonies
- BraR/MRV: 6 colonies
- RpaR/MRV: 7 colonies
- MRV (neg): 3 colonies
- Success! Pick 2 colonies from plates 1-4 for streak plate and liquid cultures (5 mL each)
Rene - check gRNA assemblies
- Minipreps from 4/20/15
- Note: ran out of time last week to grow up cultures. Stored at 4C until this week, grew for ~5 hours and then miniprepped w/ Sigma GenElute kit (elution vol. = 75 μL)
- Check w/ BpiI/XbaI
- gRNA51, 1
- gRNA51, 2
- gRNA52, 1
- gRNA52, 2
- gRNA53, 1
- gRNA53, 2 (Note: Accidentally mixed 6 w/ 7 during pellet resuspension!)
- gRNA54 (Note: Accidentally mixed 6 w/ 7 during pellet resuspension!)
- gRNA55, 1
- gRNA55, 2
- gRNA56, 1
- gRNA56, 2
- pX330 (ctrl)
Reagent
|
Rxn 1-11
|
Mix(x13)
|
Expected pX330 = ~8335, 195 gRNA+pX330 = 8530
|
15 μL/lane; 1% agarose; Ladder
|
DNA |
3.0 |
---
|
10X buffer |
1.5 |
19.5
|
BpiI |
0.5 |
6.5
|
XbaI |
0.5 |
6.5
|
dH2O |
9.5 |
123.5
|
|
15.0 |
|
- Conclusion
- Gel inconclusive, expected sizes not seen for pX330 control
- Proceed with sequencing
Sample |
OD260 |
260/280 |
ng/μL
|
1. gRNA51-1 |
0.233 |
1.928 |
232.82
|
2. gRNA51-2 |
0.112 |
1.901 |
112.471
|
3. gRNA52-1 |
0.203 |
1.901 |
203.39
|
4. gRNA52-2 |
0.237 |
1.923 |
236.79
|
5. gRNA53-1 |
0.212 |
1.903 |
212.34
|
6. gRNA55-1 |
0.233 |
1.906 |
233.19
|
7. gRNA55-2 |
0.242 |
1.896 |
242.43
|
8. gRNA55-1 |
0.217 |
1.895 |
217.49
|
9. gRNA55-2 |
0.264 |
1.896 |
264.27
|
Sequencing list:
- g51-1, f (P139)
- g51-1, r (P140)
- g51-2, f
- g51-2, r
- g52-1, f
- g52-1, r
- g52-2, f
- g52-2, r
- g53-1, f
- g53-1, r
- g55-1, f
- g55-1, r
- g55-2, f
- g55-2, r
- g56-1, f
- g56-1, r
- g56-2, f
- g56-2, r
- Cultures for new minipreps
- 6 & 7 were accidentally mixed
- Set up 5 mL cultures for gRNA53-2 and gRNA54
LCR Development
- Final round of Phusion PCR reactions
Phusion PCR:
- MV10 + pol
- MV10 + pol
- MV10 no pol (neg ctrl to see how much of band is template)
Reagent
|
Rxn1,2
|
Rxn3
|
Expected: 1,2. MV10 = 5191 3. No product
|
5 μL/lane; 1% agarose; Ladder
|
DNA(clean linear) |
1.0 μL |
1.0 μL
|
10 μM F primer |
1.0 |
1.0
|
10 μM R primer |
1.0 |
1.0
|
10 mM dNTPs |
1.0 |
1.0
|
Phusion Pol. |
0.5 |
---
|
5X HF buffer |
10.0 |
10.0
|
dH2O |
35.5 |
36.0
|
|
50.0 |
50.0
|
Program: Phusion (block A)
- 98°C, 3 min
- 35x[98°C, 10 sec; 65°C, 30 sec; 72°C, 2.5 min]
- 72°C, 10 min
- 4°C ∞
Conclusion
- Lane 3 shows product. Must have accidentally added Phusion pol.
- Desired band not any brighter than reaction done at 60°C (see 04/17/15).
- Use reactions from 4/17/15 instead of these for LCR.
Phusion PCR:
- Gal4DB-mCh - plasmid
- "
- ATF2 - plasmid template
- "
- ATF2 - 1:10 iPSC cDNA template
- "
Reagent
|
Rxn1-4
|
Rxn5,6
|
Expected: 5,6. Gal4DB-mCh = 1170 7-10. ATF2 = 906
|
5 μL/lane; 1% agarose; Ladder
|
DNA(clean linear) |
0.5 μL |
2.0 μL
|
10 μM F primer |
1.0 |
1.0
|
10 μM R primer |
1.0 |
1.0
|
10 mM dNTPs |
1.0 |
1.0
|
Phusion Pol. |
0.5 |
0.5
|
5X HF buffer |
10.0 |
10.0
|
dH2O |
36.0 |
34.5
|
|
50.0 |
50.0
|
Program: Phusion (block B)
- 98°C, 3 min
- 35x[98°C, 10 sec; 55°C, 30 sec; 72°C, 60 sec]
- 72°C, 10 min
- 4°C ∞
Conclusion
- Gal4DB-mCh - much better than previous reactions. Purify these (combined) and use for LCR
- ATF2 - rxns w/ plasmid template look awesome! cDNA templates give a spurious band. Use plasmid PCR's (combined, purified) for LCR.
|