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Team:HokkaidoU Japan/Notebook/August18
From 2010.igem.org
(Difference between revisions)
(→Digestion) |
(→エタ沈) |
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→Incubated at 37C for 60 min | →Incubated at 37C for 60 min | ||
| - | == | + | ==Ethanol Precipication== |
| - | * 2 | + | * Added 2 uL of sodium acetate(3M) |
| - | * 44 | + | * Added 44 uL of Ethanol |
| - | * | + | * Transfered to 1.5 mL tube and frozen with liquid nitrogen |
| - | * | + | * Melted and centrifuged at 15,996 rpm, 4C for 5 min |
| - | * | + | * Transfered supernatant to another tube |
| - | * 100 | + | ** Driven by precaution centrifuged the supernatant and discarded it`s supernatant |
| - | * | + | * Rinsed the tube walls with 100 uL of 70% Ethanol and centrifuged at 15,996 rpm, 4C for 5 min |
| - | * | + | * Discarded the supertenant and dried via vacuum desiccator |
| + | * Melted in 5 uL of TE | ||
==電気泳動== | ==電気泳動== | ||
Revision as of 16:42, 21 September 2010
since 13 Jul 2010
since 1 Aug 2010
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 toLambda/Hind III (ng/10 uL) | ng/uL | ratio | size (bp) | (ng) required | (uL) used |
|---|---|---|---|---|---|---|
| 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 |
| double 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
- ncubated at 37C for 15~20 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(3M)
- Added 44 uL of Ethanol
- Transfered to 1.5 mL tube and frozen with liquid nitrogen
- Melted and centrifuged at 15,996 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 15,996 rpm, 4C for 5 min
- Discarded the supertenant and dried via vacuum desiccator
- Melted in 5 uL of TE
電気泳動
- TEで溶かしたDNA Solution 1 uLに6x SB 1 uLを加えた
- 最初にとった上清も同じ操作をした
| Lane | DNA |
| 2 | TSUDA Marker 1 |
| 3 | 上清 |
| 4 | DNA solution |
- DNA solutionにしっかり回収されていた
