06/23/15
- Cas-tone - Assemblies: H3 parts into MV11
- Rene - CRISPR library, continue dirty PCR
Cas-tone Assemblies
- Do-over - use inserts with improved revrse primer (eliminate frame-shift)
- H3-1-40_MV11: H3-1-40/(X/N)/129 + MV11/(S/N)/9506
- H3-1-60_MV11: H3-1-60/(X/N)/189 + "
- H3-1-80_MV11: H3-1-80/(X/N)/250 + "
- H3-1-116_MV11: H3-1-116/(X/N)/373 + "
- H3-1-136_MV11: H3-1-136/(X/N)/433 + "
- Digest of vector (Fermentas FD)
Reagent
|
Volume
|
|
DNA (plasmid) |
20.0
|
10x buffer |
3.0
|
SpeI |
1.0
|
NotI |
1.0
|
dH2O |
5.0
|
|
30 μL --> 37°C/ ~15 min.
|
- Gel purification
- Sigma GenElute kit. Elute & back elute w/ 25 μL elution sln.
- DpnI digest & clean-up of PCR products
- Add 1 μL DpnI to each PCR rxn. Incubate @ 37°C/ 5 min.
- Use the Qiagen Qiaquick PCR Purification kit (cat. no. 28104 ) to purify DNA.
- Elute & back-elute with 30 μL of elution solution.
- Measure [DNA] for vector and inserts
Sample |
OD260 |
260/280 |
ng/μL
|
1. MV11 (S/N) |
0.019 |
1.596 |
19.4
|
2. H3-1-40 PCR |
0.024 |
1.908 |
24.2
|
3. H3-1-60 PCR |
0.041 |
1.984 |
40.8
|
4. H3-1-80 PCR |
0.044 |
1.917 |
43.8
|
5. H3-1-116 PCR |
0.067 |
1.865 |
67.1
|
6. H3-1-136 PCR |
0.072 |
1.901 |
72.4
|
Reagent
|
Volume
|
DNA (500 ng) |
up to 15.0 μL
|
10X FD buffer |
2.0
|
FD XbaI |
1.0
|
FD NotI |
1.0
|
Roche SAP |
1.0
|
dH2O |
x μL
|
|
20.0
|
Thermal cycler: LabNet OptiMax "AnOlig shrt"
- 37°C, 10 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, ∞
- Final concentration = 25 ng/μL.
- Ligations
- > 2:1 ratio calculations...
- 433 bp largest insert / 9506 bp vector * 2 * 50 ng vector = 4.5 ng
- 129 bp smallest insert / 9506 bp vector * 2 * 50 ng vector = 1.4 ng
Reagent
|
Rxn1-5
|
Rxn6
|
Insert DNA |
0.5 |
---
|
Vector DNA |
2.5 |
2.5
|
2x lgn buf (Roche) |
5.0 |
5.0
|
T4 ligase (NEB) |
1.0 |
1.0
|
dH2O |
1.0 |
1.5
|
|
10.0 μL |
10.0 μL
|
RESULTS (5/22/15)
- Success! Ligation plates have 15-20 colonies, neg. ctrl has 1 colony
- Pick 2 colonies from each plate for 5 mL cultures and streaks
|