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 reverse 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)
- MV11 - SpeI/NotI
Reagent
|
Volume
|
30 μL/lane, 1% agarose; Ladder
|
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 (inserts)
- 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 (6/24/15)
- SUCCESS! Plate 6 (neg ctrl) has 7 colonies; Plates 1-5 have 30 - 50 colonies
- Pick two clones from each for 5 mL cultures/ minipreps
Rene - CRISPR PCR library PCR
- Part 1 - GoTaq "Dirty" PCR on liquid mini-cultures, 600 bp amplicons; primers P163/P215
- Part 2 - Nested SYBR real time PCR & melt curves on (1) mini cultures, (2) amplicons from GoTaq
- Part 1 - GoTaq "Dirty" PCR on liquid mini-cultures, 600 bp amplicons
- Clone labeling notes, changed from last time:
- L34 = Luc14 cells treated with gRNA034
- G34 = Gal4EED/luc cells treated with gRNA034
- 001, 002, 003 = clone no.
- A01, A02, etc. = well position in dish or spot on agar array
1-96. L34_097_A01 - L34_192_H12 (full plate)
Reagent
|
Rxn1-24
|
Mix (x104)
|
Expected: PCR insert/ pJET = ~600 bp 1. L34_097_A01 2. L34_109_B01 3. L34_121_C01 4. L34_133_D01 5. L34_145_E01 6. L34_157_F01 7. L34_169_G01 8. L34_181_H01
|
5 μL/lane, 1% agarose; Ladder
|
Template (culture) |
2.0 |
---
|
10 uM fwd primer |
1.0 |
104.0
|
10 uM rev primer |
1.0 |
104.0
|
2x GoTaq (clear) |
12.5 |
1300.0
|
dH2O |
8.5 |
884.0
|
|
25.0 μL
|
- Note: Master Mix Multiplier (for one 96-well plate) = 96 + 1 extra 8-well column = 104
- Aliquot 200 μL into 8 tubes in 8-tube strip
- Use multichannel to aliquot 23 μL to 96-well plate, 1 column at a time
- Refill the 8-tube strip as needed with enough to fill remaining columns
PCR - Labnet: "GoTaq35KAH"
- 98°C, 10 min
- 35x[95°C, 10 sec; 60°C, 10 sec; 72°C, 20 sec]
- 72°C, 3 min
- 4°C ∞
CONCLUSIONS
|