04/23/15
- Ryan - Receiver cloning stage 2
- Line item 2
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
Reagent
|
Volume
|
DNA (500 ng plasmid) |
up to 16.0 μL
|
10x buffer |
2.0
|
EcoRI |
1.0
|
XbaI |
1.0
|
dH2O |
###
|
|
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 (500 ng)
|
phosphatase |
1.0
|
dH2O |
---
|
|
20 μL --> 37°C/ 10 min.; 75°C/ 2 min.; [final] = ~25 ng/μL
|
Measure Vector conc's
- Ligation/Digestion Reactions
- For an insert = ~200 and vector = ~4000, need 5 ng insert for 2:1 insert:vector ratio
- Insert is mostly old vector (3000 bp), ~6% is insert
- So, use at least 84 ng of the E/X dephos insert DNA
|
1 |
2 |
|
Insert DNA |
### |
--- |
|
Vector DNA |
### |
### |
|
2x lgn buf (Roche) |
### |
### |
|
T4 ligase (NEB) |
1.0 |
1.0 |
|
dH2O |
### |
### |
|
|
# μL |
# μL |
|
|