05/14/15
- Ryan - minipreps & digests
- Rene - PCR cloning trial 2
- Cas-tone - assembly stage 1
Minipreps
- Check with E/KpnI digests
- pAub-AubR/MRV
- pBja-BjaR/MRV
- pBra-BraR/MRV
- pRpa-RpaR/MRV
| Reagent
|
Volume
|
Expected:
1,2. pAub/AubR/MRV = 4240
3,4. pBja/BjaR/MRV = 4099
5,6. pBra/BraR/MRV = 4191
7,8. pRpa/RpaR/MRV = 4116
9. Empty MRV = 2230, 971
np pro. Reg/MRV = ~4000, 971
|
15 μL/lane; 1% agarose; Ladder
|
| DNA(plasmid) |
2.0 μL
|
| 10X buffer |
1.5
|
| EcoRI |
1.0
|
| KpnI |
1.0
|
| dH2O |
9.5
|
| |
15 μL
|
--> 37°C/ ~10 min.
PCR cloning trial 2
- 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 (2.0 μL insert, from 5/13/15)
- Gal4EED/luc + CRISPR g034 = 630 (2.0 μL insert, from 5/13/15)
- Luc14 + CRISPR g034; size = 630 (4.0 μL insert)
- Gal4EED/luc + CRISPR g034 = 630 (4.0 μL insert)
| Reagent
|
Volume
|
| 2x reaction buffer |
10.0
|
| PCR product (>31.5 ng) |
4.0
|
| pJET1.2/blunt vector |
1.0
|
| T4 ligase |
1.0
|
| dH2O |
4.0
|
| |
20.0 μL
|
- Incubate the ligation mixture at room temperature (22°C) for 5 min.
- Transform 50 μL DH5α-turbo with 10 μL ligation reaction. Follow long protocol (42°C heat shock/ recovery).
- Plate on 100 μg/mL amp.
RESULTS (5/15/15)
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
- 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
- Design primers for H2A, H2B, H3, H4, and tails
- Insert Kozak-histone parts (X/N) into dCas9 (S/N)
- STAGE 4 - gRNA
- Put pre-existing gRNA (from luc experiment) into pSPgRNA
Minipreps & Digests
- (prep 2) pcDNA-dCas9-VP64 - X/HindIII = 6450, 2345, 1017
- (" 1) pSPgRNA - BbsI = 3201, 22
No XbaI or HindIII sites
- (" 3) pHR-SFFV-dCas9-BFP-KRAB - X/HindIII = 4955, 3127, 2606, 840, 587, 563, 556, 535, 255, 107, 24, 12
- (" 4) pMSCV-LTR-dCas9-VP64-BFP - X/HindIII = 5517, 3638, 927, 733, 554
Low copy, use 5.0 uL
- (" 5) mEmerald-H2A-10 - X/HindIII = 866, 311, 26
- (" 6) mEmerald-H3-23 - X/HindIII = 788, 446, 27
- (" 7) mEmerald-H4-23 - X/HindIII = 788, 346, 27
- (from Vi's project) H2B-GFP (pEGFP-N1) - X/HindIII = 3944, 1163
| Reagent
|
Rxns 1,3,5-8
|
Rxn 2
|
Rxn 4
|
Expected:
1. BB 1 = size
2. BB2 = size
|
|
| DNA(plasmid) |
2.0 |
2.0 |
5.0
|
| 10X buffer (FD) |
1.5 |
1.5 |
1.5
|
| XbaI/BbsI (FD) |
1.0 |
1.0 |
1.0
|
| HindIII (NEB HF) |
1.0 |
--- |
1.0
|
| dH2O |
9.5 |
10.5 |
4.5
|
| |
15 μL
|
--> 37°C/ ~10 min.
Assemblies
- CMV-dCas-VP64_pcDNA: CMV/(X/SacII)/588 + pcDNA-dCas9-VP64/(S/SacII)/9056
- Digest & gel purify vector (Fermentas FD)
- pcDNA-dCas9-VP64, S/SacII
| Reagent
|
Volumes
|
|
| DNA (plasmid) |
up to 25 μL |
500 ng |
15
|
| 10x buffer |
3.0
|
| enzyme 1 |
1.0
|
| enzyme 2 |
1.0
|
| dH2O |
---
|
| |
30 μL --> 37°C/ ~30 min.
|
- Gel purify: Sigma GenElute; elute & back elute w/ 25 μL elution sln.
- Measure conc.
| Sample |
OD260 |
260/280 |
ng/μL
|
| 1. pcDNA-dCas9-VP64 (S/SacII) |
--- |
--- |
---
|
- Digest & dephos insert (PCR clip & clone method)
- XbaI-CMV-SpeI-NotI-SacII PCR (clean), X/SacII (see 05/11/15
- Digest & dephos PCR fragments
| Reagent
|
Volume
|
| DNA (500 ng) |
2.7 μL
|
| 10X buffer (FD clear) |
2.0
|
| SacII |
1.0
|
| XbaI |
1.0
|
| SAP (Roche) |
1.0
|
| dH2O |
5.0
|
| |
20.0
|
- LabNet OptiMax Thermocycler: 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, ∞
| 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 |
|
|
Karmella's BioBrick Cloning
