Team:HokkaidoU Japan/Notebook/August27

From 2010.igem.org

(Difference between revisions)
(New page: {{Template:HokkaidoU_Japan}})
 
(12 intermediate revisions not shown)
Line 1: Line 1:
{{Template:HokkaidoU_Japan}}
{{Template:HokkaidoU_Japan}}
 +
<div class="linkbar"><div class="prev">[[Team:HokkaidoU_Japan/Notebook/August26|August 26]]</div>[[Team:HokkaidoU_Japan/Notebook|Notebook]]<div class="next">[[Team:HokkaidoU_Japan/Notebook/August30|August 30]]</div></div>
 +
 +
=Transfer [[Team:HokkaidoU_Japan/Materials_And_Methods#BioBricks|1-1A]] on pSB1A10 to pSB1C3=
 +
1.estimated concentrations of pSB1C3 and [[Team:HokkaidoU_Japan/Materials_And_Methods#BioBricks|1-1A]] which was amplified by miniprep before
 +
 +
2.mixed solutions according to the table below
 +
 +
 +
<div style="float:left;">
 +
{|style="text-align: center;" class="protocol"
 +
|-
 +
!Reagent
 +
!Amount
 +
|-
 +
|pSB1C3
 +
|5 uL
 +
|-
 +
|DW
 +
|75 uL
 +
|-
 +
|10x M Buffer
 +
|10 uL
 +
|-
 +
|0.1%BSA
 +
|10 uL
 +
|-
 +
|EcoRI
 +
|5 uL
 +
|-
 +
|PstI
 +
|5 uL
 +
|-
 +
|style="border-top:1px solid #996"|'''Total'''
 +
|style="border-top:1px solid #996"|'''100 uL'''
 +
|}
 +
</div>
 +
<div style="float:left; margin-left:50px;">
 +
{|style="text-align: center;" class="protocol"
 +
|-
 +
!Reagent
 +
!Amount
 +
|-
 +
|1-1A
 +
|8 uL
 +
|-
 +
|DW
 +
|6 uL
 +
|-
 +
|10x M Buffer
 +
|2 uL
 +
|-
 +
|0.1%BSA
 +
|2 uL
 +
|-
 +
|EcoRI
 +
|1 uL
 +
|-
 +
|PstI
 +
|1 uL
 +
|-
 +
|style="border-top:1px solid #996"|'''Total'''
 +
|style="border-top:1px solid #996"|'''20 uL'''
 +
|}
 +
</div>
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
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
 +
 +
{|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
 +
|}
 +
 +
 +
1.estimated concentrations of Heat Sensor,RBS,RFP and Double Terminator
 +
 +
2.mixed solutions according to the table below
 +
 +
<div style="float:left;">
 +
{|style="text-align: center; margin-left:20px" class="protocol"
 +
|-
 +
!Reagent
 +
!Amount
 +
|-
 +
| DW
 +
| 13.5 uL
 +
|-
 +
| 10x M buffer
 +
| 5 uL
 +
|-
 +
| 0.1%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'''
 +
|}
 +
</div>
 +
<div style="float:left;">
 +
{|style="text-align: center; margin-left:20px" class="protocol"
 +
|-
 +
!Reagent
 +
!Amount
 +
|-
 +
| DW
 +
| 34.9 uL
 +
|-
 +
| 10x M buffer
 +
| 5 uL
 +
|-
 +
| 0.1%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'''
 +
|}
 +
</div>
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
<div style="float:left;">
 +
{|style="text-align: center; margin-left:20px" class="protocol"
 +
|-
 +
!Reagent
 +
!Amount
 +
|-
 +
| DW
 +
| 25.5 uL
 +
|-
 +
| 10x M buffer
 +
| 5 uL
 +
|-
 +
| 0.1%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'''
 +
|}
 +
</div>
 +
<div style="float:left;">
 +
{|style="text-align: center; margin-left:20px" class="protocol"
 +
|-
 +
!Reagent
 +
!Amount
 +
|-
 +
| DW
 +
| 34 uL
 +
|-
 +
| 10x M buffer
 +
| 5 uL
 +
|-
 +
| 0.1%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'''
 +
|}
 +
</div>

Latest revision as of 01:13, 28 October 2010

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
0.1%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
0.1%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
0.1%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
0.1%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
0.1%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
0.1%BSA 5 uL
XbaI 1 uL
PstI 2 uL
Double Terminator 3 uL
Total 50 uL