Team:HokkaidoU Japan/Notebook/August18
From 2010.igem.org
RBS digestion: Revenge
Reagent | Amount |
---|---|
1-2M | 10 uL |
DW | 4 uL |
10x M Buffer | 2 uL |
BSA | 2 uL |
Xba I | 1 uL |
Pst I | 1 uL |
Total | 20 uL |
->Incubated at 37C for 60 min
- Added 4 uL of 6x Sample Buffer making it total of 12 uL per lane and electrophoresed
->Extracted for gel
->Electrophoresed 10 uL of Extracted DNA
- Used TSUDA Marker 1
Failure
- After better inspection of Kit specs it came to lite that retreavel rate for 50 bp and less is 26%
- RBS is just quite small when cut
Ligation
Preparation of DNA Solution for Ligation
Part | By comparison toλ/Hind III (ng/10 uL) | ng/uL | ratio | size(bp) | required(ng) | used(uL) |
---|---|---|---|---|---|---|
Vector | 50 ng/10 uL | 5 ng/uL | 1 | 2996 bp | 10 ng | 2 uL |
RFP | 250 ng/10 uL | 25 ng/uL | 2 | 700 bp | 7 ng | 0.3 uL |
terminator | 5 ng/10 uL | 0.5 ng/uL | 2 | 200 bp | 2 ng | 4 uL |
Total | 6.3 uL |
Ligation and Transformation
Reagent | Amount |
---|---|
DNA solution | 6.3 uL |
Ligation solution | 6.3 uL |
T4 ligase | 1 uL |
Total | 13.6 uL |
- Incubated at 16C for 30 min
- Transformation: Added all to 50 uL of competent cell
- Incubated at 0C for 30 min
- Heat shocked at 42C for 60 sec
- 5 min on ice
- Added 100 uL of LB
- Incubated at 37C for 120 min
- Spread onto the LBC plate
- Incubated at 37C for 20 hrs
RBS Retry
Digestion
Reagent | Amount |
---|---|
(RBS)1-2M | 10 uL |
DW | 4 uL |
10x M Buffer | 2 uL |
BSA | 2 uL |
Xba I | 1 uL |
Pst I | 1 uL |
Total | 20 uL |
->Incubated at 37C for 60 min
Ethanol Precipication
- Added 2 uL of sodium acetate(3 M)
- Added 44 uL of Ethanol
- Transfered to 1.5 mL tube and frozen with liquid nitrogen
- Melted and centrifuged at 15000 rpm, 4C for 5 min
- Transfered supernatant to another tube
- Driven by precaution centrifuged the supernatant and discarded it's supernatant
- Rinsed the tube walls with 100 uL of 70% Ethanol and centrifuged at 15000 rpm, 4C for 5 min
- Discarded the supertenant and dried via vacuum desiccator
- Melted in 5 uL of TE
Electrophoresis
- Added 1 uL of 6x SB to DNA Solution of 1 uL
- Did the same to supernatant retreaved for precaution
Lane | DNA |
2 | TSUDA Marker 1 |
3 | supernatant |
4 | DNA solution |
- DNA solution band is visible
- Looks OK