05/21/15
- Cas-tone - Assemblies, H3's + MV11
Cas-tone Project
- STAGE 1 - pcDNA-dCas9-VP64 vector re-design
- DONE - 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)
- DONE PCR-amplify histone parts as XbaI-Kozak-histone part-a-NotI. Note: Do no use Kozak BioBrick, since this will add extra amino acids onto conserved histone N-terminal tail. Instead, use the Kozak-like seq. from the Addgene plasmid, +3 extra Kozak bases (included in primer)
- STAGE 3 - Cas-fusion
- Insert Kozak-histone parts (X/N) into dCas9 plasmid "MV11" (S/N)
- STAGE 4 - gRNA
- Put pre-existing gRNA (from luc experiment) into pSPgRNA
Assemblies
- H3-1-40_MV11: H3-1-40/(X/N)/size + MV11/(S/N)/9506
- H3-1-60_MV11: 5' part/(a/b)/size + MV11/(S/N)/9506
- Digest of vector (Fermentas FD)
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.
|
- Gel purification
- Sigma GenElute kit. Elute & back elute w/ 25 μL elution sln.
- DpnI digest & clean-up of PCR products
- Add 1 μL DpnI to each PCR rxn. Incubate @ 37°C/ 5 min.
- Use the Qiagen Qiaquick PCR Purification kit (cat. no. 28104 ) to purify DNA.
- Elute with 30 μL of elution solution.
- Measure [DNA] for vector and inserts
Sample |
OD260 |
260/280 |
ng/μL
|
1. Insert 1 PCR |
### |
### |
###
|
2. Insert 2 PCR |
### |
### |
###
|
3. etc. |
### |
### |
###
|
- Set up restriction digest/ phosphatase reactions as shown in the table below
- FD = Fermentase FastDigest buffer/ enzyme
- SAP = shrimp alkaline phosphatase
Reagent
|
Volume
|
DNA (500 ng) |
up to 15.0 μL
|
10X FD buffer |
2.0
|
FD EcoRI |
1.0
|
FD XbaI |
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, ∞
Your insert is now ready to use. The final concentration is 25 ng/μL.
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
|
|