# Team:HokkaidoU Japan/Notebook/August27

(Difference between revisions)
 Revision as of 12:40, 13 October 2010 (view source)Sho N (Talk | contribs)← Older edit Revision as of 13:35, 13 October 2010 (view source)Sho N (Talk | contribs) Newer edit → Line 4: Line 4: =Transfer 1-1A on pSB1A10 to pSB1C3= =Transfer 1-1A on pSB1A10 to pSB1C3= - 1.estimated concentration of pSB1C3 and 1-1A which was amplified by miniprep before + 1.estimated concentrations of pSB1C3 and 1-1A which was amplified by miniprep before 2.mixed solutions according to the table below 2.mixed solutions according to the table below Line 117: Line 117: *Parts Information *Parts Information - Heat Sensor + {|border="1" style="margin-left: 20px;" class="protocol" + |- + !Name + !Biobrick Name + !Well + !Length + |- + | Heat Sensor + | BBa_K098995 + | 3-1E + | 937 bp + |- + | RBS + | BBa_B0034 + | 1-2M + | 12 bp + |- + | RFP + | BBa_E1010 + | 1-18F + | 681 bp + |- + | Double Terminator + | BBa_0015 + | 1-23L + | 129 bp + |} - BBa_K098995 3-1E 937bp - RBS + 1.estimated concentrations of Heat Sensor,RBS,RFP and Double Terminator - BBa_B0034 1-2M 12bp + 2.mixed solutions according to the table below - RFP + {|style="text-align: center; margin-left:20px" class="protocol" - + |- - BBa_E1010 1-18F 681bp + !Reagent - + !Amount - Double Terminator + |- - + | DW - BBa_0015 1-23L 129bp + | 13.5 uL + |- + | 10x M buffer + | 5 uL + |- + | 1%BSA + | 5 uL + |- + | EcoRI + | 3 uL + |- + | SpeI + | 1 uL + |- + | Heat Sensor + | 22.5 uL + |- + |style="border-top:1px solid #996;"|'''Total''' + |style="border-top:1px solid #996;"|'''50 uL''' + |} + {|style="text-align: center; margin-left:20px" class="protocol" + |- + !Reagent + !Amount + |- + | DW + | 34.9 uL + |- + | 10x M buffer + | 5 uL + |- + | 1%BSA + | 5 uL + |- + | XbaI + | 1 uL + |- + | PstI + | 2 uL + |- + | RBS + | 34.9 uL + |- + |style="border-top:1px solid #996;"|'''Total''' + |style="border-top:1px solid #996;"|'''50 uL''' + |} + {|style="text-align: center; margin-left:20px" class="protocol" + |- + !Reagent + !Amount + |- + | DW + | 25.5 uL + |- + | 10x M buffer + | 5 uL + |- + | 1%BSA + | 5 uL + |- + | EcoRI + | 3 uL + |- + | SpeI + | 1 uL + |- + | RFP + | 10.5 uL + |- + |style="border-top:1px solid #996;"|'''Total''' + |style="border-top:1px solid #996;"|'''50 uL''' + |} + {|style="text-align: center; margin-left:20px" class="protocol" + |- + !Reagent + !Amount + |- + | DW + | 34 uL + |- + | 10x M buffer + | 5 uL + |- + | 1%BSA + | 5 uL + |- + | XbaI + | 1 uL + |- + | PstI + | 2 uL + |- + | Double Terminator + | 3 uL + |- + |style="border-top:1px solid #996;"|'''Total''' + |style="border-top:1px solid #996;"|'''50 uL''' + |}

# Transfer 1-1A on pSB1A10 to pSB1C3

1.estimated concentrations of pSB1C3 and 1-1A which was amplified by miniprep before

2.mixed solutions according to the table below

Reagent Amount
pSB1C3 5 uL
DW 75 uL
10x M Buffer 10 uL
1%BSA 10 uL
EcoRI 5 uL
PstI 5 uL
Total 100 uL
Reagent Amount
1-1A 8 uL
DW 6 uL
10x M Buffer 2 uL
1%BSA 2 uL
EcoRI 1 uL
PstI 1 uL
Total 20 uL

3.incubated at 37C for 60min

4.purified the two samples

5.added 250 uL 100% EtOH to each samples

6.centrifuged at 4C, 15000rpm for 10 min

8.put the samples into desiccator

9.added 10 uL DW to pSB1C3 and 2 uL DW to 1-1A

10.mixed 1 uL pSB1C3 and 2 uL 1-1A

11.added 3 uL ligation solution and 0.5 uL T4 ligase

12.transfered the sample to a tube of 500 uL

13.incubated at 16C for 30 min

->For not putting 3M CH3COONa into the solutions,we stopped operation up to this.

# Preparation of 3 Piece Ligation

• Parts Information
Name Biobrick Name Well Length
Heat Sensor BBa_K098995 3-1E 937 bp
RBS BBa_B0034 1-2M 12 bp
RFP BBa_E1010 1-18F 681 bp
Double Terminator BBa_0015 1-23L 129 bp

1.estimated concentrations of Heat Sensor,RBS,RFP and Double Terminator

2.mixed solutions according to the table below

Reagent Amount
DW 13.5 uL
10x M buffer 5 uL
1%BSA 5 uL
EcoRI 3 uL
SpeI 1 uL
Heat Sensor 22.5 uL
Total 50 uL
Reagent Amount
DW 34.9 uL
10x M buffer 5 uL
1%BSA 5 uL
XbaI 1 uL
PstI 2 uL
RBS 34.9 uL
Total 50 uL
Reagent Amount
DW 25.5 uL
10x M buffer 5 uL
1%BSA 5 uL
EcoRI 3 uL
SpeI 1 uL
RFP 10.5 uL
Total 50 uL
Reagent Amount
DW 34 uL
10x M buffer 5 uL
1%BSA 5 uL
XbaI 1 uL
PstI 2 uL
Double Terminator 3 uL
Total 50 uL