04/16/15
- LCR Development - PCR MV10 bacbone
- gRNA cloning (Rene)
LCR Development
- Try PCR on linearized vector
Reagent
|
Volume
|
DNA(plasmid) |
5.0 μL
|
10X buffer |
3.0
|
XbaI |
2.0
|
dH2O |
20.0
|
|
30 μL --> 37°C/ ~30 min.
|
Measure conc.
- DNA purified with Sigma PCR clean-up kit
- Eluted& back-eluted with 30 μL elution sln.
Sample |
OD260 |
260/280 |
ng/μL
|
1. MV10 XbaI |
0.017 |
1.47 |
17.24
|
Phusion PCR
- 1.0 μL template, HF buffer
- 2.0 μL template, HF buffer
- 1.0 μL template, GC buffer
- 2.0 μL template, GC buffer
Reagent
|
Rxn1,3
|
Rxn2,4
|
Expected: MV10 band = 5191
|
10 μL/lane, 1% agarose; Ladder
|
Template |
1.0 |
2.0
|
10 uM fwd primer |
1.0 |
1.0
|
10 uM rev primer |
1.0 |
1.0
|
10 mM dNTPs |
1.0 |
1.0
|
Phusion pol. |
0.5 |
0.5
|
5x buffer (HF/GC) |
5.0 |
5.0
|
dH2O |
40.5 |
39.5
|
|
50.0 |
50.0
|
Program: Phusion (block B)
- 98°C, 3 min
- 35x[98°C, 10 sec; 60°C, 30 sec; 72°C, 30 sec]
- 72°C, 10 min
- 4°C ∞
Conclusion
- Not ideal, proceed with caution
Measure conc.
- PCR 1 & 2 combined, purified with Sigma PCR clean-up kit
- Eluted& back-eluted with 30 μL elution sln.
Sample |
OD260 |
260/280 |
ng/μL
|
1. 5'p-MV10 PCR |
### |
### |
###
|
gRNA cloning - Rene
- Follow the "Oligo annealing and cloning into backbone vectors - NEW" protocol
Phosphorylate and anneal oligo pairs
- g051 T/B
- g052 T/B
- g053 T/B
- g054 T/B
- g055 T/B
- g056 T/B
Reagent
|
Rxn
|
Mix (7x)
|
100 μM Oligo 1 |
1.0 |
---
|
100 μM Oligo 2 |
1.0 |
---
|
10x T4 Lign buf (NEB) |
1.0 |
7.0
|
T4 PNK (NEB) |
0.5 |
3.5
|
dH2O |
6.5 |
45.5
|
|
10.0
|
LabNet OptiMax Thermocycler (AnOlig RD)
- 37°C, 30 min
- 95°C, 5 min
- Ramp down to 25°C, 5°C/1 min. [90/1 min, 85/1 min, 80/1 min, ... 25°C/1 min]
- 25°C, ∞
Dilute the product(s) 1:250
- Add 2 μL product to 498 μL dH2O
Digestion/ Ligation Reactions (Vector / dsOligo Insert)
- pX330 / g051
- g052 T/B
- g053 T/B
- g054 T/B
- g055 T/B
- g056 T/B
Reagent
|
Rxn
|
100 ng Vector |
##
|
1:250 dsOligo |
2.0
|
10x FD buf (NEB) |
2.0
|
10 mM DTT |
1.0
|
10 mM ATP |
1.0
|
FastDigest BbsI |
1.0
|
T7 DNA ligase |
0.5
|
dH2O |
##
|
|
20.0
|
Bio-Rad Thermocycler
- 6x [37°C, 5 min; 23°C, 5 min]
- 4°C, ∞
Transformation(s)
- 2.0 μL ligation + 50 μL DH5α-turbo
- Plate on 100 μg/mL amp
|