05/18/15
- Ben - assembly: HPK-CFP into homology arms
- Cas-tone - modification #1
- Rene - PCR cloning trial 3
Ben - assembly: HPK-CFP into homology arms
- DBN006 / DBN003 f1 (Tm 71.6), DBN002 r1 (Tm 59.4)
- same
Reagent
|
Volume
|
Mix (x2)
|
Expected: 1,2. DBN006 = 1972
|
5 μL/lane; 1% agarose; Ladder
|
DNA(plasmid) |
0.5 μL |
1.0
|
10 μM F primer |
1.0 |
2.0
|
10 μM R primer |
1.0 |
2.0
|
10 mM dNTPs |
1.0 |
2.0
|
Phusion Pol. |
0.5 |
1.0
|
5X HF buffer |
10.0 |
20.0
|
dH2O |
36.0 |
72.0
|
|
50.0 |
|
Program: Phusion (block B)
- 98°C, 3 min
- 35x[98°C, 10 sec; 57°C, 30 sec; 72°C, 30 sec]
- 72°C, 10 min
- 4°C ∞
Conclusion
- Failed - Tm too high. The binding site for one of the primers is actually 49°C. Redesign this primer.
Cas-tone Project
- STAGE 1 - pcDNA-dCas9-VP64 vector re-design
- Knock-out VP64 from C-terminus - replace AscI-VP64:HA:STOP-EcoRI-XbaI with AscI-HA:STOP-XbaI dsOligo
- DONE - Knock-out FLAG from N-terminus - replace SpeI-CMV:3xFLAG:NLS-SacII with PCR-amplified (SpeI)/XbaI-CMV-SpeI-NotI-SacII; use reverse primer to add SacII
- STAGE 2 - Histone parts
- DONE - Order primers (annotated in Benchling)
- PCR-clone histone parts as XbaI-Kozak-histone part-a-NotI. Note: Do no use Kozak BioBrick, since this will introduce unnatural amino acids onto conserved histone N-terminal tail
- STAGE 3 - Cas-fusion
- Insert Kozak-histone parts (X/N) into dCas9 (S/N)
- STAGE 4 - gRNA
- Put pre-existing gRNA (from luc experiment) into pSPgRNA
Assembly
- CMV-dCas-HA: Cas47107_Mod1/(dsOligo)/50 + CMV-dCas-VP64/(AscI/XbaI)/9453
Reagent
|
Volume
|
30 μL/lane, 1% agarose; Ladder
|
DNA (plasmid) |
15.0
|
10x buffer |
3.0
|
AscI |
1.0
|
XbaI |
1.0
|
dH2O |
10.0
|
|
30 μL
|
--> 37°C/ ~30 min.
- Gel purify
- Sigma GenElute kit; elute & back-elute w/ 25 μL elution sln.
Sample |
OD260 |
260/280 |
ng/μL
|
1. CMV-dCas-VP64/(AscI/XbaI) |
0.041 |
2.005 |
40.5
|
1. CMV-dCas-VP64 miniprep |
0.19 |
1.93 |
189.5
|
1. Luc14 g034 |
0.035 |
1.97 |
34.8
|
1. Gal4EED g034 |
0.039 |
1.867 |
38.7
|
- Phosphorylate and anneal oligo pair
- Cas47107_Mod1 top/btm
Reagent
|
Rxn
|
Mix (2x)
|
100 μM Oligo 1 |
1.0 |
2.0
|
100 μM Oligo 2 |
1.0 |
2.0
|
10x T4 Lign buf (NEB) |
1.0 |
2.0
|
T4 PNK (NEB) |
0.5 |
1.0
|
dH2O |
6.5 |
13.0
|
|
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
- Ligations
- 50 bp insert / 9453 bp vector * 2 * 100 ng vector = 1.06 ng insert
- Cas47107_Mod1(1:250 dsOligo) + CMV-dCas-VP64(AscI/XbaI)
- CMV-dCas-VP64(AscI/XbaI)
Reagent
|
Rxn1
|
Rxn2
|
Insert DNA |
2.0 |
---
|
Vector DNA |
1.5 |
1.5
|
2x lgn buf (Roche) |
5.0 |
5.0
|
T4 ligase (NEB) |
1.0 |
1.0
|
dH2O |
0.5 |
2.5
|
|
10.0 μL |
10.0 μL
|
RESULTS (5/19/15)
- Success! ~100 colonies on ligation plate, 6 on neg ctrl.
- Pick two colonies for 5mL cultures
Rene - PCR cloning trial 3
- This time use Roche ligation buffer and NEB T4 ligase
- Use the CloneJET PCR Cloning Kit - MAN0012707 CloneJET PCR Cloning (manual)
- pJET1.2/blunt vector is 5'-phosphorylated and accepts blunt products (Phusion PCR)
- All common laboratory E. coli strains can be directly transformed with the ligation product
- Re-circularized vector expresses toxic enzyme, no blue/white screening needed
- Optimal insert : vector (0.05 pmol ends) ratio is 3:1; Calculation: length of insert DNA x 0.05 = ng insert to use
- Ligations & transformations - CloneJET PCR Cloning Kit
- Luc14 + CRISPR g034; size = 630 (use 31.5 ng)
- Gal4EED/luc + CRISPR g034; size = 630 (use 31.5 ng)
Reagent
|
Volume
|
2x Roche lig buf |
5.0
|
PCR product (>31.5 ng) |
1.0
|
pJET1.2/blunt vector |
1.0
|
NEB T4 ligase |
1.0
|
dH2O |
2.0
|
|
10.0 μL
|
- Incubate the ligation mixture at room temperature (22°C) for 5 min.
- Transform 50 μL DH5α-turbo with 10 μL ligation reaction. Follow short protocol.
RESULTS (5/19/15)
- Success! ~200 colonies on each plate. Pick for 96-well "mass culture" plates
|