05/07/15
- Ryan - Dig/Lig reaction do-over
Ryan - Dig/Lig reaction do-over
- This time include neg ctrls for each reaction
- Dig/Lig reactions
- 2:1 ratio calculation: ~150 bp insert / ~4000 bp vector * 2 * 100 = 3.75 ng insert
- pAub-PCR(E/S dp) + AubR/MRV
- AubR/MRV
- pBja-PCR(E/S dp) + BjaR/MRV
- BjaR/MRV
- pBra-PCR(E/S dp) + BraR/MRV
- BraR/MRV
- pRpa-PCR(E/S dp) + RpaR/MRV
- RpaR/MRV
| Reagent
|
Rxn
|
Mix (x9)
|
| 100 ng Vector |
### (up to 13.5) |
---
|
| insert |
1.0 |
---
|
| 10x FD buf |
2.0 |
18.0
|
| 10 mM DTT |
1.0 |
9.0
|
| 10 mM ATP |
1.0 |
9.0
|
| FastDigest BbsI/BpiI |
1.0 |
9.0
|
| Roche T4 DNA ligase |
0.5 |
4.5
|
| dH2O |
### |
---
|
| |
20.0
|
--> Pipette 5.5 μL master mix into each PCR tube
--> Add 1.0 μL insert into each tube
--> Add 100 ng vector DNA
--> Add x μL dH2O = 13.5 - vector volume
--> Mix by flicking
LabNet OptiMax Thermocycler: BbsI Dig/Lig
- 6x [37°C, 5 min; 23°C, 5 min]
- 4°C, ∞
Transformation(s)
- Thaw 2 tubes chem competent DH5α-turbo on ice
- Transfer 10.0 μL each ligation (1/2 rxn.) to fresh sterile 0.5 mL tube
- Add 50 μL DH5α-turbo; pipette up-and-down 3x GENTLY
- Incubate 5 min. on ice
- Plate on 100 μg/mL amp
RESULTS (5/08/15)
- ~25 colonies on each ligation plate, and same number on negative controls
- Rene has observed something similar for dig/lig.
- Pick grow up liquid cultures for two colonies from each plate on Monday
|
Karmella's BioBrick Cloning
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