04/21/15
- Ryan - regulator assemblies
- Rene - check gRNA assemblies; digests & sequencing
- LCR Development - Phusion PCR
Ryan - Receiver plasmids, assembly
- Stage 1 - insert Regulator ORFS
- DONE - 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
- Clean up PCR products
- Zymo clean and concentrator
- Elute w/ 25 μL dH2O
- AubR/MRV: AubR-PCR(E/X)/851 + MRV(E/X)/3201
- BjaR/MRV: BjaR-PCR(E/X)/776 + MRV(E/X)/3201
- BraR/MRV: BraR-PCR(E/X)/776 + MRV(E/X)/3201
- RpaR/MRV: RpaR-PCR(E/X)/779 + MRV(E/X)/3201
- AubR, 851 bp
- BjaR, 776 bp
- BraR, 776 bp
- RpaR, 779 bp
- Modular Receiver Vector (MRV)
Reagent
|
Rxn 1-4
|
Rxn 5
|
DNA |
10.0 |
5.0
|
10X buffer |
3.0 |
3.0
|
EcoRI |
1.0 |
1.0
|
XbaI |
1.0 |
1.0
|
dH2O |
15.0 |
20.0
|
|
30.0 |
30.0
|
- Column clean-up
- Zymo clean and concentrator
Sample |
OD260 |
260/280 |
ng/μL
|
1. AubR-PCR (E/X) |
-0.005 |
5.875 |
-5.001
|
2. BjaR-PCR (E/X) |
-0.005 |
9 |
-4.788
|
3. BraR-PCR (E/X) |
-0.005 |
7.143 |
-5.291
|
4. RpaR-PCR (E/X) |
-0.003 |
29 |
-3.052
|
5. MRV (E/X) |
0.007 |
1.388 |
7.11
|
- Note: unsure why concentrations so low. Proceed with ligations anyway, see what happens.
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 |
|
Rene - check gRNA assemblies
- Minipreps from 4/20/15
- Note: ran out of time last week to grow up cultures. Stored at 4C until this week, grew for ~5 hours and then miniprepped w/ Sigma GenElute kit (elution vol. = 75 μL)
- Check w/ BpiI/XbaI
- gRNA51, 1
- gRNA51, 2
- gRNA52, 1
- gRNA52, 2
- gRNA53, 1
- gRNA53, 2 (Note: Accidentally mixed 6 w/ 7 during pellet resuspension!)
- gRNA54 (Note: Accidentally mixed 6 w/ 7 during pellet resuspension!)
- gRNA55, 1
- gRNA55, 2
- gRNA56, 1
- gRNA56, 2
- pX330 (ctrl)
Reagent
|
Rxn 1-11
|
Mix(x13)
|
Expected pX330 = ~8335, 195 gRNA+pX330 = 8530
|
Hover label
|
DNA |
3.0 |
---
|
10X buffer |
1.5 |
19.5
|
BpiI |
0.5 |
6.5
|
XbaI |
0.5 |
6.5
|
dH2O |
9.5 |
123.5
|
|
15.0 |
|
|