Team:Chiba/jk/β/week 3
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Week 3
31 August, 2008
- Transformation
- Competent Cells : XL10G
- ---> We saved these plates with a refrigerator.
Sample No 1~3 4 Sample DNA 12 10 PstⅠ 0.2 0.2 XbaⅠ 0.2 - SpeⅠ - 0.4 Buffer 2 - 1.5 Buffer 3 2 - BSA 2 1.5 dH2O 3.6 1.4 TOTAL 20 15
- --->(1/9)Gel Check
- (30/8)--->Mini prep
- --->Digestion test
- Plac+RBS+RhlI Sample No.2~5
- BBa_T9002[7]①、②
Sample Single Digesiton 2~5 Double Digestion 2~5 Single Digestion T9002① Single Digesiton T9002② Double Digestion T9002①② Sample DNA 1 3 3 1 3 XbaⅠ 0.1 0.1 0.1 0.1 0.1 SpeⅠ - 0.1 - - 0.1 Buffer 2 0.9 0.8 0.9 0.9 0.8 BSA 1 1 1 1 1 dH2O 7 5 5 7 5 TOTAL 10 10 10 10 10
- --->Gel Check
1 September, 2008
- (31/8)--->Gel Check
Sample No 1~3 4 Sample DNA 20 15 Loading Dye 4 3 TOTAL 24 18 - From left;
- insert-1(I9026)
- insert-2(I9030)
- From left;
- insert-3(S03154)
- vector-4(R0010)
- --->Gel extract
- --->zymo
- insert-1(I9026) -> 7μL
- insert-2(I9030) -> 7μL
- insert-3(S03154) -> 7μL
- vector-4(R0010) -> 15μL
- --->SAP
- vector-4(R0010)
- --->Zymo
- vector-4(R0010) -> 20μL
- --->Gel Check
- --->Ligation
Sample No (1) (2) (3) (4) (5) insert-1(I9026) 3 - 3 - - insert-2(I9030) - 3 - 3 - vector-4(R0010) 3 3 - - 3 ligase 1 1 1 1 1 Buffer 1 1 1 1 1 dH2O 2 2 5 5 5 TOTAL 10 10 10 10 10
- --->Transformation
- Competent cells : XL10GOLD 30μL
- Transformed the following and grew on new ampicillin plates.
- --->(2/9) Colony PCR
- Competent cells : BW ΔFliC 40μL
- Transformed the following and grew on new ampicillin plates.
- --->(2/9)Liquid Culture
2 September, 2008
- (31/8)--->Gel Check
Sample DNA 1 Loading Dye 1 dH2O 4 TOTAL 6 - From left;
- I9026 -> OK, 100/μL
- I9030 -> OK, 50ng/μL
- S03154 -> OK, 30ng/μL (too low for the ligation:1/9 )
- From left;
- (1/9)---> Colony PCR
DNA Template 1 dNTP mix 5 Foward Primer 0.3 Reverse Primer 0.3 DNA polymerase TAQ 0.5 Thermopol Buffer 3 dH2O 20.5 TOTAL 30μL
- 95℃,5min -> ( 95℃,1min -> 52℃,1min -> 72℃,1min )・・・25cycles -> 72℃,10min -> 6℃
--->Gel CheckSample DNA 1 Loading Dye 1 dH2O 4 TOTAL 6 - From left;
- Plac+RBS+RhlI+LVA
- R1 -> OK
- R2 -> Bad
- R3~R7 -> OK
- R8 -> Bad
- From left;
- From left;
- Plac+RBS+CinI+LVA
- C1,C2 -> OK
- C3 -> Bad
- C4~C6 -> OK
- From left;
- Plac+RBS+CinI+LVA
- C7,C8 -> OK
- BBa_F2620[23](2007):Positive control -> OK
- --->(3/9)Mini prep
(1/9)--->Liquid Culture- Cultured the following cells (2mL LB-Amp, at 37℃, 7 hours)
--->(3/9)Phenotype test
--->(4/9)Mini prep
3 September, 2008
- --->Gel Check
- From left;
- BBa_K084010[37](C1,C2,C4~C8) -> OK
- --->Digestion test
Digestion Single Double Sample DNA 1 3 XbaⅠ 0.1 0.1 PstⅠ 0.1 0.1 Buffer 2 0.9 - Buffer 3 - -0.8 BSA 1 1 dH2O 7 5 TOTAL 10μL 10μL
- --->Gel Check
- (2/9)--->Phenotype-test
- MIX
Sample No. 1 2 3 4 5 6 7 8 9 10 11 L1 2mL - - - - - - - - - - L2 - 2mL - - - - - - - - - L3 - - 2mL - - - - - - - - L4 - - - 2mL - - - - - - - R1 - - - - 1mL - - - - - - R2 - - - - - 1mL - - - - - R3 - - - - - - 1mL - - - - R4 - - - - - - - 1mL - - - BBa_S03154[48] - - - - - - - - 2mL - - AHL(100μM) - - - - - - - - - - 1μL BBa_T9002[49] 2mL 2mL 2mL 2mL 1mL 1mL 1mL 1mL 2mL 1mL 1mL IPTG(100μM) 1μL 1μL 1μL 1μL 1μL 1μL 1μL 1μL - - - - Incubated for 8hours at 37 degrees
- Spindown (max rpm, 3 min)
- The measurement of the intensity GFP by Masahiro Tominaga
Sample No 1 2 3 4 5 6 7 8 9 10 11 the intensity GFP + + + + + + + ++ + +++ - 4 September, 2008
- --->Gel Check
- --->[Team:Chiba/protocol/digestion|Digestion test]]
Sample Single Digesiton Double Digestion Sample DNA 1 2 EcoRⅠ 0.05 0.05 PstⅠ - 0.05 Buffer 3 1 1 BSA 1 1 dH2O 6.95 5.9 TOTAL 10 10
- --->Gel Check
Sample DNA 10 Loading Dye 2 TOTAL 12
- From left;
- Single Digestion
- ladder
- Double digestion
- Transformation
- Competent cells : BW ΔFliC
- Transformed the following and grew on new ampicillin plates.
- --->(5/9)Liquid Culture
5 September, 2008
- (4/9)--->Liquid Culture
- Cultured the following cells (2mL LB-Amp, at 37℃)
- --->Phenotype test
- MIX
Sample No. 1 2 3 4 5 6 7 8 9 10 11 r3 1mL - - - - - - - - - - r4 - 1mL - - - - - - - - - r6 - - 1mL - - - - - - - - r7 - - - 1mL - - - - - - - c1 - - - - 1mL - - - - - - c2 - - - - - 1mL - - - - - c6 - - - - - - 1mL - - - - c7 - - - - - - - 1mL - - - BBa_S03154[80] - - - - - - - - 1mL - - AHL(100μM) - - - - - - - - - - 1μL BBa_T9002[81] 1mL 1mL 1mL 1mL 1mL 1mL 1mL 1mL 1mL 1mL 1mL IPTG(100μM) 1μL 1μL 1μL 1μL 1μL 1μL 1μL 1μL - - - - Incubated for 8hours at 37 degrees
- The measurement of the intensity GFP by KEIKOU-READER
Sample No 1 2 3 4 5 6 7 8 9 10 11 the intensity GFP (Frist run) 15.21 16.44 16.34 16.63 9.720 9.703 9.685 9.874 19.95 16.59 71.04 the intensity GFP (Second run) 17.85 19.19 19.28 19.14 9.981 10.16 10.14 10.41 79.53 17.61 23.94
- Spindown (max rpm, 3 min)
- The measurement of the intensity GFP by Masahiro Tominaga (UV 312nm)