04/23/15
- Ryan - Receiver cloning stage 2
- Rene - sequencing for g053-2, g054
Ryan - Receiver plasmids, assembly
- 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
- Cut & dephos promoters with E/X
- Insert promoters(E/X) 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
Sample |
OD260 |
260/280 |
ng/μL
|
1. AubR/MRV |
0.258 |
1.906 |
257.7
|
2. BjaR/MRV |
0.177 |
1.904 |
177.3
|
3. RpaR/MRV |
0.246 |
1.896 |
246.2
|
- Digests (Fermentas FD)
- E/X
- Inserts are gBlocks = 2.0 ng/μL
Reagent
|
Volume
|
DNA (20 ng) |
10.0 μL
|
10x buffer |
2.0
|
EcoRI |
1.0
|
XbaI |
1.0
|
dH2O |
6.0
|
|
20.0 μL --> 37°C/ ~15 min.
|
- Dephosphorylation (Roche)
- Assuming that shrimp alkaline phosphatase works in FastDigest buffer conditions Link
Reagent
|
Volume
|
DNA (digest) |
20.0 μL (20 ng)
|
phosphatase |
1.0
|
dH2O |
---
|
|
20 μL
|
--> 37°C/ 10 min.; 75°C/ 2 min.; [final] = ~1 ng/μL
- Digestion/Ligation Reactions (borrowed from Rene's gRNA/Cas9 assembly procedure)
- Inserts are gBlocks = ~2ng/μL
- For an insert = ~200 and vector = ~4000, need 5 ng insert for 2:1 insert:vector(50 ng) ratio
- pAub/AubR/MRV: pAub(E/Xdp)/153 + AubR/MRV(BbsI)/4087
- pBja/BjaR/MRV: pBja(E/Xdp)/122 + BjaR/MRV(BbsI)/3977
- pRpa/RpaR/MRV: pRpa(E/Xdp)/136 + RpaR/MRV(BbsI)/3980
- Note: BraR cloning was not successful, omit from this round of assembly
|
1 |
2 |
3
|
Insert DNA |
5.0 |
5.0 |
5.0
|
Vector DNA |
0.2 |
0.3 |
0.2
|
10x FD buf |
2.0 |
2.0 |
2.0
|
10 mM DTT |
1.0 |
1.0 |
1.0
|
10 mM ATP |
1.0 |
1.0 |
1.0
|
FD BbsI/BpiI |
1.0 |
1.0 |
1.0
|
T4 ligase (Roche) |
1.0 |
1.0 |
1.0
|
dH2O |
8.8 |
8.7 |
8.8
|
|
20.0 μL |
20.0 μL |
20.0 μL |
|
Thermal cycler: Labnet - BbsI Dig/Lig
- 6x[37°C, 5 min.; 23°C 5 min.]
- 4°C, ∞
Rene - sequencing
- g53-2, f (P139) - Confirmed
- g53-2, r (P140) - low Phred 20 score
- g54, f (P139) - Confirmed
- g54, r (P140) - low Phred 20 score
|