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
|
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/MV10-1 |
0.273 |
1.948 |
273.0
|
| 2. KAH87/MV10-2 |
0.341 |
1.940 |
341.5
|
| 3. KAH87/MV10-3 |
0.319 |
1.937 |
318.5
|
| 4. KAH87/MV10-4 |
0.248 |
1.948 |
248.1
|
Conclusions:
- Success! Clone #1,2,4 have forward insertion
- Discard - clone #3, reverse insertion
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
|
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 |
0.021 |
1.707 |
20.8
|
| 2. pBjaR PCR |
0.018 |
1.851 |
18.3
|
| 3. pBraR PCR |
0.026 |
1.807 |
25.5
|
| 4. pRpaR PCR |
0.018 |
1.720 |
17.8
|
- Digests (Fermentas FD)
- Use clear FD buffer (no dye)
| Reagent
|
Volume
|
| DNA (250 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)
- Add 1.0 SAP (Roche) to each rxn.
- 37°C/ 10 min.; 75°C/ 2 min.; [final] = 12.5 ng/μL
- Dig/Lig reactions
- 2:1 ratio calculation: ~150 bp insert / ~4000 bp vector * 2 * 100 = 3.75 ng insert
- pAub-PCR(E/S dp) + AubR/MRV
- pBja-PCR(E/S dp) + BjaR/MRV
- pBra-PCR(E/S dp) + BraR/MRV
- pRpa-PCR(E/S dp) + RpaR/MRV
| Reagent
|
Rxn
|
Mix (x5)
|
| 100 ng Vector |
### (up to 13.5) |
---
|
| insert |
1.0 |
---
|
| 10x FD buf |
2.0 |
10.0
|
| 10 mM DTT |
1.0 |
5.0
|
| 10 mM ATP |
1.0 |
5.0
|
| FastDigest BbsI/BpiI |
1.0 |
5.0
|
| Roche T4 DNA ligase |
0.5 |
2.5
|
| dH2O |
### |
---
|
| |
20.0
|
--> Pipette 5.5 μL master mix into each PCR tube
--> Add 1.0 μL insert into each tube
--> Add 100 ng vector DNA
--> Add x μL dH2O = 13.5 - vector volume
--> Mix by flicking
LabNet OptiMax Thermocycler: BbsI Dig/Lig
- 6x [37°C, 5 min; 23°C, 5 min]
- 4°C, ∞
Transformation(s)
- Thaw 2 tubes chem competent DH5α-turbo on ice
- Transfer 10.0 μL each ligation to fresh sterile 0.5 mL tube
- Add 50 μL DH5α-turbo; pipette up-and-down 3x GENTLY
- Incubate 5 min. on ice
- Plate on 100 μg/mL amp
- Split ligations in 1/2, do rapid protocol
RESULTS (5/06/15)
- Success! ~50 colonies on the four assembly plates, ~10 colonies on a negative control (ligation mix, zero DNA)
- Streak plate and 5 mL cultures for two colonies from each assembly plate
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:3xFLAG:NLS-SacII with PCR-amplified (SpeI)/XbaI-CMV-SpeI-NotI-SacII; use reverse primer to add SacII
- STAGE 2 - Histone parts
- 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
- Order primers
- 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
- Application
- Cas and gRNA plasmids will be co-transformed
- Analyze Cas protein via Western blot
Knock-out VP64 from C-terminus
- Design and order oligos for AscI-HA:STOP-EcoRI dsOligo
- Cas47107_VP64ko top1 - 5'-CGCGCCATTAACTACCCGTACGACGTTCCGGACTACGCTTCTTGAGCGGCCGT
- Cas47107_VP64ko btm - 5'-ctagACGGCCGCTCAAGAAGCGTAGTCCGGAACGTCGTACGGGTAGTTAATGG
Knock-out FLAG from N-terminus
- Design and order oligos to PCR-amplify CMV
- XbaI-CMV f1 - 5'-CCTTTCTAGAGTTGACATTGATTATTGGCTAG
- SacII-NS-CMV r1 - 5'-CATTCCGCGGGCGGCCGCTACTAGTGAGCTCTGC
|
Karmella's BioBrick Cloning
