04/16/15
- LCR Development - PCR MV10 backbone
- 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
|
Mistake: Buffer volume should be 10/rxn, no 5! Consider re-run
Program: Phusion (block B)
- 98°C, 3 min
- 35x[98°C, 10 sec; 60°C, 30 sec; 72°C, 2.5 min]
- 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 |
0.008 |
1.28 |
7.77
|
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
- pX330 / g052
- pX330 / g053
- pX330 / g054
- pX330 / g055
- pX330 / g056
| Reagent
|
Rxn
|
Mix (x7)
|
| 100 ng Vector |
0.5 |
3.5
|
| 1:250 dsOligo |
2.0 |
---
|
| 10x FD buf |
2.0 |
14.0
|
| 10 mM DTT |
1.0 |
7.0
|
| 10 mM ATP |
1.0 |
7.0
|
| FastDigest BbsI |
1.0 |
7.0
|
| T4 DNA ligase |
0.5 |
3.5
|
| dH2O |
12.0 |
84.0
|
| |
20.0
|
LabNet OptiMax Thermocycler: BbsI Dig/Lig
- 6x [37°C, 5 min; 23°C, 5 min]
- 4°C, ∞
Transformation(s)
- 10.0 μL ligation + 50 μL DH5α-turbo
- Plate on 100 μg/mL amp
- Split ligations in 1/2, do rapid protocl for 1 set, long protocol for other set
RESULTS (4/17/15)
- Success! Paltes 1-3, 5, 6 had ~5 colonies. Plate 4 had just one.
- Pick two colonies from each (except plate 4, pick 1)
- Make streak plate & set up 5 mL cultures
|
Karmella's BioBrick Cloning
