05/05/15
- KAH87/MV10 - minipreps (5x 5mL)
- Ryan - clone-in Receiver promoters
- Cas-tone Project - plan assembly strategy, order primers
Minipreps
- Sigma GenElute kit
- Elute w/ 75 μL elution sln.
- Check with NotI/XbaI digests
Reagent
|
Volume
|
Expected: KAH87 fwd/MV10 = ~6150, 36, 28 KAH87 rev/MV10 = ~5100, 1089, 28 MV10 = ~5100, 36, 28
|
Hover name 15 μL/lane; 1% agarose; Ladder
|
DNA(plasmid) |
2.0 μL
|
10X buffer |
1.5
|
NotI |
1.0
|
XbaI |
1.0
|
dH2O |
9.5
|
|
15 μL --> 37°C/ ~15 min.
|
Sample |
OD260 |
260/280 |
ng/μL
|
1. KAH87/MV10 |
--- |
--- |
---
|
2. Digested part (c/d) |
--- |
--- |
---
|
Ryan - PCR & Dig/Lig (promoter insert trial #3)
Ryan - Receiver plasmids, assembly - REVISED STRATEGY (PCR promoters)
- Stage 1 - insert Regulator ORFS
- Phusion PCR-amplify ORFs (add 5'-E, 3'-X cut sites)
- Insert ORF(E/X) into Vector (E/X). see Vector plasmid map
- Stage 2 - insert Promoters
- Phusion PCR-amplify promoter inserts (new primers)
- Cut promoters with E/S & dephos
- Insert promoters(E/S) into Vector/Regulator (BbsI) via Lig/Dig cycling
Assemblies
- pAub/AubR/MRV: pAub(E/Xdp)/153 + AubR/MRV(BbsI)/4087
- pBja/BjaR/MRV: pBja(E/Xdp)/122 + BjaR/MRV(BbsI)/3977
- pBra/BraR/MRV: pBra(E/Xdp)/214 + BraR/MRV(BbsI)/3977
- pRpa/RpaR/MRV: pRpa(E/Xdp)/136 + RpaR/MRV(BbsI)/3980
- Vector concentrations (ng/μL)
- AubR/MRV, 257.7
- BjaR/MRV, 177.3
- BraR/MRV, 465.4
- RpaR/MRV, 246.2
- Phusion PCR-amplify promoters
- pAubR, EcoRI fwd, SpeI rev
- pBjaR, EcoRI fwd, SpeI rev
- pBraR, EcoRI fwd, SpeI rev
- pRpaR, EcoRI fwd, SpeI rev
Reagent
|
Vol
|
Mix (x5)
|
Expected: 1. pAubR = 153 2. pBjaR = 122 3. pBraR = 214 4. pRpaR = 136
|
Hover name 5 μL/lane; 1% agarose; Ladder
|
gBlock DNA (2 ng/μL) |
0.5 |
---
|
10 μM EcoRI fwd |
1.0 |
5.0
|
10 μM SpeI rev |
1.0 |
5.0
|
10 mM dNTPs |
1.0 |
5.0
|
5x HF buffer |
10.0 |
50.0
|
Phusion Pol. |
0.5 |
2.5
|
dH2O |
36.0 |
180.0
|
|
50.0 μL |
|
Thermal cycler: Bio-Rad - Phusion
- 98°C 3 min.
- 30x[98°C, 10 sec; 66°C 30 sec; 72°C 30 sec]
- 72°C 3 min.
- 4°C, ∞
- Clean up PCR
- Qiagen PCR purification kit
- Elute w/ 30 μL elution buffer
Sample |
OD260 |
260/280 |
ng/μL
|
1. pAubR PCR |
--- |
--- |
---
|
2. pBjaR PCR |
--- |
--- |
---
|
3. pBraR PCR |
--- |
--- |
---
|
4. pRpaR PCR |
--- |
--- |
---
|
- Digests (Fermentas FD)
- Use clear FD buffer (no dye)
Reagent
|
Volume
|
DNA (500 ng PCR) |
up to 16.0
|
10x clear FD buffer |
2.0
|
EcoRI |
1.0
|
SpeI |
1.0
|
dH2O |
---
|
|
20.0 μL
|
--> 37°C/ ~15 min.
- Dephosphorylation (Roche)
Reagent
|
Volume
|
DNA (clean digest) |
up to 17 μL (500 ng)
|
10x buffer d.p. |
2.0
|
phosphatase |
1.0
|
dH2O |
---
|
|
20 μL --> 37°C/ 10 min.; 75°C/ 2 min.; [final] = 25 ng/μL
|
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
- Build CMV:Kozak/V0120
- Knock-out FLAG from N-terminus - replace SpeI-CMV:FLAG:NLS-SacII with PCR-amplified (SpeI)/XbaI-CMV:Kozak-SpeI-NotI-SacII; use reverse primer to add SacII
- STAGE 2 - histone parts
- Design histone components with RFC23 and SacII 3' in V0120
- Order primers
- STAGE 3 - Cas assembly
- Replace SpeI-CMV:NLS:3xFLAG-SacII with SpeI-CMV:histonepart-SacII
- STAGE 4 - gRNA
- Put pre-existing gRNA (from luc experiment) into pSPgRNA
- Application
- Cas and gRNA plasmids will be co-transformed
- Analyze Cas protein via Western blot
Redesign dCas: design and order oligos for AscI-HA:STOP-EcoRI dsOligo
- Cas47107_VP64ko top1 - 5'-CGCGCCATTAACTACCCGTACGACGTTCCGGACTACGCTTCTTGAGCGGCCGT
- Cas47107_VP64ko btm - 5'-ctagACGGCCGCTCAAGAAGCGTAGTCCGGAACGTCGTACGGGTAGTTAATGG
Design histone components with RFC23 and SacII 3' in V0120
- RFC23prefix-H3_full:linker-SacII-RFC23suffix
- RFC23prefix-H3_1-40:linker-SacII-RFC23suffix
Assemblies
- BioBrick name: 5' part/(a/b)/size + 3' part/(c/d)/size
- BioBrick name: 5' part/(a/b)/size + 3' part/(c/d)/size
Reagent
|
Volume
|
|
DNA (plasmid) |
up to 25 μL
|
10x buffer |
3.0
|
enzyme 1 |
1.0
|
enzyme 2 |
1.0
|
dH2O |
---
|
|
30 μL --> 37°C/ ~30 min.
|
Sample |
OD260 |
260/280 |
ng/μL
|
1. Digested part (a/b) |
--- |
--- |
---
|
2. Digested part (c/d) |
--- |
--- |
---
|
- Dephosphorylation (Roche)
Reagent
|
Volume
|
DNA (clean digest) |
up to 17 μL (500 ng)
|
10x buffer d.p. |
2.0
|
phosphatase |
1.0
|
dH2O |
---
|
|
20 μL --> 37°C/ 10 min.; 75°C/ 2 min.; [final] = 25 ng/μL
|
Ligation |
Plate results (lig : neg crtl) mm/dd/yy
|
1. insert(a/b)/size, ## ng + vector(c/d)/size, ## ng |
new BioBrick #:1 (Pick #)
|
2. vector(c/d)/ ## ng |
|
|
1 |
2 |
|
Insert DNA |
### |
--- |
|
Vector DNA |
### |
### |
|
2x lgn buf (Roche) |
### |
### |
|
T4 ligase (NEB) |
1.0 |
1.0 |
|
dH2O |
### |
### |
|
|
# μL |
# μL |
|
Oligo annealing
- New BB 1
- New BB 2
DNA (oligos, 100 μM) |
up to 18 μL (3 μL ea.)
|
10x annealing buffer |
2.0
|
dH2O |
---
|
|
20 μL --> 100°C (water bath)/ 5 min.; Cool to R.T. overnight
|
|