Team:HokkaidoU Japan/Notebook/August27

From 2010.igem.org

(Difference between revisions)
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'''
 +
|}

Revision as of 13:35, 13 October 2010

Notebook

ラオリ英語添削お願い

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

7.discarded supernatant

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
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