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24 August 2008 <|> 26 August 2008
Laboratory work
Team:Input
BBa_J22136(insert check済み)のグリセロールストックづくり
プレートに生えたコロニーをpickして、試験管で2ml培養(LB-Amp,37℃,12h)
培養した試験管から180μl,LB-Ampを880μlとって、OD=0.495にした。
| Liquid culture(OD=0.495) |
0.67μl |
| 60%Glycerol |
0.33μl |
| 20%Glycerol Stock |
1.00ml |
Team:Communication
- (24/8)--->Digestion
- Double Digestion:BBa_J04500(2007)
- Double Digesiton:BBa_R0010(2007)
- Single Digestion:BBa_R0010(2007)
- BBa_R0010(2007)
| Sample No. |
1 | 2 | 3 | 4 |
| Sample DNA |
5 | 5 | 1 | 1 |
| EcoRⅠ |
0.5 | 0.5 | - | - |
| SpeⅠ |
0.5 | 0.5 | - | - |
| Buffer 1 |
1 | 1 | - | - |
| Buffer 3 |
- | - | 1 | - |
| BSA |
1 | 1 | - | - |
| dH2O |
2 | 2 | 7.5 | - |
| TOTAL |
10 | 10 | 10 | 1 |
- --->Gel Check
|
| Sample No. |
1~3 | 4 |
| Sample DNA |
10 | 1 |
| Loading Dye |
2 | 1 |
| dH2O |
- | 4 |
| TOTAL |
12 | 6 |
- From right;
- Double Digestion:BBa_J04500(2007) -> OK
- Double Digesiton:BBa_R0010(2007) -> ?(low)
- Single Digestion:BBa_R0010(2007) -> ?(low)
- BBa_R0010(2007) -> OK(low)
|
--->Digestion
(Double Digestion)
- BBa_C0178
- BBa_C0170
- BBa_J04500(2007)
| Sample No. |
1 | 2 | 3 |
| Sample DNA |
33ng/μl×60μl | 33ng/μl×60μl | 100ng/μl×20μl |
| PstⅠ |
2 | 2 | 2 |
| XbaⅠ |
2 | 2 | - |
| SpeⅠ |
- | - | 2 |
| Buffer 2 |
- | - | 5 |
| Buffer 3 |
10 | 10 | - |
| BSA |
10 | 10 | 5 |
| dH2O |
16 | 16 | 16 |
| TOTAL |
100 | 100 | 52 |
Team:Output
PCR
| Sample No. |
① | ② | ③ | ④ | ⑤ |
| DNA tamplate |
1 | 1 | 1 | 1 | >1 |
| FW primer |
5(Venus_YFP_fwd) | 5(LacZ_fwd) | 5(LacZ_fwd) | 5(rLUC_fwd) | 5(fLuc_fwd) |
| VR primer |
5(VR) | 5(LacZ_rev) | 5(LacZ_rev) | 5(VR) | 5(VR) |
| dNTPmix |
10 | 10 | 10 | 10 | 10 |
| Thermo pol buffer |
10 | 10 | 10 | 10 | 10 |
| DNA pol(VENT) |
1 | 1 | 1 | 1 | 1 |
| dH2O |
68 | 68 | 68 | 68 | 68 |
| TOTAL |
100 | 100 | 100 | 100 | 100 |
-->Gel Check
| Sample No. |
① | ② | ③ | ④ | ⑤ |
| DNA tamplate |
1 | 1 | 1 | 1 | >1 |
| loading Dye |
1 | 1 | 1 | 1 | 1 |
| dH2O |
4 | 4 | 4 | 4 | 4 |
| TOTAL |
6 | 6 | 6 | 6 | 6 |
Mini prep
Transformation
- Venus
- PUC19
- pGFPUV
- pGEX Venus YFP
- Membren Venus YFP
PCR
| Sample No. |
① |
| DNA tamplate |
1 |
| FW primer(Venus) |
5 |
| VR primer |
5 |
| dNTPmix |
10 |
| Thermo pol buffer |
10 |
| DNA pol(VENT) |
1 |
| dH2O |
68 |
| TOTAL |
100 |
"
