Team:HokkaidoU Japan/Notebook/September6

From 2010.igem.org

(Difference between revisions)
(New page: {{Template:HokkaidoU_Japan}})
 
(10 intermediate revisions not shown)
Line 1: Line 1:
-
{{Template:HokkaidoU_Japan}}
+
{{Template:HokkaidoU_Japan}}<div class="linkbar"><div class="prev">[[Team:HokkaidoU_Japan/Notebook/September3|September 3]]</div>[[Team:HokkaidoU_Japan/Notebook|Notebook]]<div class="next">[[Team:HokkaidoU_Japan/Notebook/September7|September 7]]</div></div>
 +
 
 +
=Ligation of pSB1C3 PCRed from [[Team:HokkaidoU_Japan/Parts#BioBricks|1-3A]] and [[Team:HokkaidoU_Japan/Parts#BioBricks|1-5A]]（RFP reporter）=
 +
 
 +
* Purified through gel extraction to remove primers
 +
* Digested as listed in the table below
 +
 
 +
{|style="text-align:center; float: left;" class="protocol"
 +
!Reagent
 +
!Amount
 +
|-
 +
|RFP ([[Team:HokkaidoU_Japan/Parts#BioBricks|1-5A]])
 +
|0.75 uL
 +
|-
 +
|DW
 +
|34.3 uL
 +
|-
 +
|0.1% BSA
 +
|5 uL
 +
|-
 +
|10x '''M''' Buffer
 +
|5 uL
 +
|-
 +
|EcoR I
 +
|2.5 uL
 +
|-
 +
|Pst I
 +
|2.5 uL
 +
|-
 +
|style="border-top:1px solid #996;"|'''Total'''
 +
|style="border-top:1px solid #996;"|'''50 uL'''
 +
|}
 +
 
 +
{|style="text-align:center; float: left;" class="protocol"
 +
!Reagent
 +
!Amount
 +
|-
 +
|pSB1C3
 +
|0.75 uL
 +
|-
 +
|DW
 +
|34.3 uL
 +
|-
 +
|0.1% BSA
 +
|5 uL
 +
|-
 +
|10x '''M''' Buffer
 +
|5 uL
 +
|-
 +
|EcoR I
 +
|2.5 uL
 +
|-
 +
|Pst I
 +
|2.5 uL
 +
|-
 +
|style="border-top:1px solid #996;"|'''Total'''
 +
|style="border-top:1px solid #996;"|'''50 uL'''
 +
|}
 +
 
 +
{|style="text-align:center; float: left;" class="protocol"
 +
!Reagent
 +
!Amount
 +
|-
 +
|RFP ([[Team:HokkaidoU_Japan/Parts#BioBricks|1-5A]])
 +
|0.75 uL
 +
|-
 +
|DW
 +
|34.3 uL
 +
|-
 +
|0.1% BSA
 +
|5 uL
 +
|-
 +
|10x '''H''' Buffer
 +
|5 uL
 +
|-
 +
|EcoR I
 +
|2.5 uL
 +
|-
 +
|Pst I
 +
|2.5 uL
 +
|-
 +
|style="border-top:1px solid #996;"|'''Total'''
 +
|style="border-top:1px solid #996;"|'''50 uL'''
 +
|}
 +
 
 +
{|style="text-align:center; float: left;" class="protocol"
 +
!Reagent
 +
!Amount
 +
|-
 +
|pSB1C3
 +
|0.75 uL
 +
|-
 +
|DW
 +
|34.3 uL
 +
|-
 +
|0.1% BSA
 +
|5 uL
 +
|-
 +
|10x '''H''' Buffer
 +
|5 uL
 +
|-
 +
|EcoR I
 +
|2.5 uL
 +
|-
 +
|Pst I
 +
|2.5 uL
 +
|-
 +
|style="border-top:1px solid #996;"|'''Total'''
 +
|style="border-top:1px solid #996;"|'''50 uL'''
 +
|}
 +
 
 +
<div style="clear:both;"></div>
 +
 
 +
* Extracted from filter with  25 uL, 50 uL DNase free water
 +
* At this point solution volumes were 22.56 ng/uL，45.12 ng/uL acordingly
 +
* Added 5 uL Mighty Mix and 0.5 uL T4 ligase to pSB1C3 2 uL, and RFP 3 uL
 +
* Ligated
 +
* [[Team:HokkaidoU_Japan/Protocols|Transformed]] to 50 uL of competent cells

Latest revision as of 08:08, 27 October 2010

Notebook

Ligation of pSB1C3 PCRed from 1-3A and 1-5A（RFP reporter）

  • Purified through gel extraction to remove primers
  • Digested as listed in the table below
Reagent Amount
RFP (1-5A) 0.75 uL
DW 34.3 uL
0.1% BSA 5 uL
10x M Buffer 5 uL
EcoR I 2.5 uL
Pst I 2.5 uL
Total 50 uL
Reagent Amount
pSB1C3 0.75 uL
DW 34.3 uL
0.1% BSA 5 uL
10x M Buffer 5 uL
EcoR I 2.5 uL
Pst I 2.5 uL
Total 50 uL
Reagent Amount
RFP (1-5A) 0.75 uL
DW 34.3 uL
0.1% BSA 5 uL
10x H Buffer 5 uL
EcoR I 2.5 uL
Pst I 2.5 uL
Total 50 uL
Reagent Amount
pSB1C3 0.75 uL
DW 34.3 uL
0.1% BSA 5 uL
10x H Buffer 5 uL
EcoR I 2.5 uL
Pst I 2.5 uL
Total 50 uL
  • Extracted from filter with 25 uL, 50 uL DNase free water
  • At this point solution volumes were 22.56 ng/uL，45.12 ng/uL acordingly
  • Added 5 uL Mighty Mix and 0.5 uL T4 ligase to pSB1C3 2 uL, and RFP 3 uL
  • Ligated
  • Transformed to 50 uL of competent cells
Retrieved from "http://2010.igem.org/Team:HokkaidoU_Japan/Notebook/September6"