05/26/15
- Ben - luc replacement donor plasmid DBN007
- Rene - CRISPR PCR library PCR trial
Ben - luc replacement donor plasmid
- DBN007_pSB1A3: DBN006(BsaI)/1972 + DBN001(BsaI)/1267+1069 (=2336)
Reagent
|
Rxn1,2
|
Expected: 1,2. DBN006 = 1972
|
5 μL/lane, 1% agarose; Ladder
|
Template |
0.2
|
10 uM fwd primer |
1.0
|
10 uM rev primer |
1.0
|
10 mM dNTPs |
1.0
|
Phusion pol. |
0.5
|
5x HF buffer |
5.0
|
dH2O |
41.3
|
|
50.0
|
Bio-Rad: "Phusion" (block #)
- 98°C, 3 min
- 30x[98°C, 10 sec; 67°C, 30 sec; 72°C, 30 sec]
- 72°C, 10 min
- 4°C ∞
CONCLUSION
- Non-specific amplification. Need to gel purify.
- Use 30 uL of each per lane for gel purification.
CONTINUED: 5/27/15
- Gel purification
- Use Sigma kit
- Elute & back-elute w/ 25 μL elution sln.
Sample |
OD260 |
260/280 |
ng/μL
|
1. DBN006 PCR |
0.002 |
-19 |
2.0
|
- Concentration is weird. Use max amt. of DNA for subsequent steps
- Digest & Dephosphorylate the Insert(s)
Reagent
|
Volume
|
DNA (500 ng) |
up to 16.0 μL
|
10X FD buffer |
2.0
|
FD BsaI |
1.0
|
Roche SAP |
1.0
|
dH2O |
x μL
|
|
20.0
|
Thermal cycler program:
Note: In the Haynes lab, use the LabNet OptiMax Thermocycler, Program "AnOlig RD"
- 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, ∞
- The final concentration is ???
- Ligations
- DBN001 2-part backbone from 05/15/15
- 2:1 ratio calc.: 1972 bp insert / 2336 bp vector * 2 * 50 = 84 ng insert
- DBN006/(BsaI)/1972 + DBN001/(BsaI)/2336
- DBN001/(BsaI)/2336
Reagent
|
Rxn1
|
Rxn2
|
Insert DNA |
6.0 |
---
|
Vector DNA |
3.0 |
3.0
|
2x lgn buf (Roche) |
10.0 |
10.0
|
T4 ligase (NEB) |
1.0 |
1.0
|
dH2O |
--- |
6.0
|
|
20.0 μL |
20.0 μL
|
- Incubate at RT/ 10 min.
- Add to 50 μL DH5α-turbo; ice/ 5min.
- Plate on 100 μg/mL amp agar
RESULTS (5/28/15)
- Success! 50 colonies on ligation plate, 5 colonies on neg. ctrl. Pick two colonies
Rene - CRISPR PCR library PCR trial
- 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:
- Lu34 = Luc14 cells treated with gRNA034
- Ga34 = Gal4EED/luc cells treated with gRNA034
- A/B = 96-well plate A or B
- A01, A02, etc. = well position in dish or spot on agar array
1-12. Luc14 g034 - Lu34_AA01 - Lu34_AA12
13-24. Gal4EED g034 Ga34_AA1 - GaA_12
Reagent
|
Rxn1-24
|
Mix (x25)
|
Expected: 1-14. PCR insert/ pJET = ~600 bp
|
10 μL/lane, 1% agarose; Ladder
|
Template (culture) |
0.5 |
---
|
10 uM fwd primer |
1.0 |
25.0
|
10 uM rev primer |
1.0 |
25.0
|
2x GoTaq (clear) |
12.5 |
312.5
|
dH2O |
10.0 |
250.0
|
|
25.0 μL
|
Labnet: "GoTaq"
- 98°C, 3 min
- 35x[95°C, 10 sec; 60°C, 10 sec; 72°C, 20 sec]
- 72°C, 3 min
- 4°C ∞
CONCLUSIONS
- Success with dirty PCR
- Continue with remainder of dirty PCR & possible SYBR PCR tomorrow
|