Team:HokkaidoU Japan/Notebook/September2

From 2010.igem.org

(Difference between revisions)
(→RFPとpSB1C3，RFPとpUC119のDigestion/ Ligation/ Transformation)
Line 4: Line 4:
{|style="text-align:center; class="protocol"
{|style="text-align:center; class="protocol"
|-
|-
-
|style="border-bottom:1px solid #000;"|DNA
+
!DNA
-
|style="border-bottom:1px solid #000;"|濃度 (ng/uL)
+
!Concentration(ng/uL)
-
|style="border-bottom:1px solid #000;"|長さ(bp)
+
!Length(bp)
-
|style="border-bottom:1px solid #000;"|量(uL)
+
!Amount(uL)
|-
|-
|RFP (1-5A)
|RFP (1-5A)
Line 27: Line 27:
==Digestion==
==Digestion==
{|style="text-align:center; float: left;" class="protocol"
{|style="text-align:center; float: left;" class="protocol"
 +
!Reagent
 +
!Amount
|-
|-
|RFP (1-5A)
|RFP (1-5A)
Line 51: Line 53:
{|style="text-align:center; float: left;" class="protocol"
{|style="text-align:center; float: left;" class="protocol"
 +
!Reagent
 +
!Amount
|-
|-
|pSB1C3
|pSB1C3
Line 81: Line 85:
[[Image:HokkaidoU Japan 20100902a.jpg|200px|right|thumb|]]
[[Image:HokkaidoU Japan 20100902a.jpg|200px|right|thumb|]]
{|class="protocol"
{|class="protocol"
 +
|'''Lane'''
 +
|'''DNA'''
|-
|-
|1
|1
Line 132: Line 138:
==Ligation==
==Ligation==
{|style="text-align:center;float: left;" class="protocol"
{|style="text-align:center;float: left;" class="protocol"
 +
!Reagent
 +
!Amount
|-
|-
|RFP
|RFP
Line 150: Line 158:
{|style="text-align:center; float: left;" class="protocol"
{|style="text-align:center; float: left;" class="protocol"
 +
!Reagent
 +
!Amount
|-
|-
|RFP
|RFP
Line 168: Line 178:
{|style="text-align:center; float: left;" class="protocol"
{|style="text-align:center; float: left;" class="protocol"
 +
!Reagent
 +
!Amount
|-
|-
|RFP
|RFP
Line 186: Line 198:
{|style="text-align:center; float: left;" class="protocol"
{|style="text-align:center; float: left;" class="protocol"
 +
!Reagent
 +
!Amount
|-
|-
|RFP
|RFP

Revision as of 06:41, 20 September 2010

Notebook

RFPとpSB1C3，RFPとpUC119のDigestion/ Ligation/ Transformation

DNA Concentration(ng/uL) Length(bp) Amount(uL)
RFP (1-5A) 80 ng/uL 1000 bp 26 uL
pSB1C3 20 ng/uL 2000 bp 35 uL
pUC119（cut済み） 10 ng/uL 3000 bp 28 uL

Digestion

Reagent Amount
RFP (1-5A) 26 uL
DW 9 uL
0.1% BSA 5 uL
10x M Buffer 5 uL
EcoR I 2 uL
Pst I 3 uL
Total 50 uL
Reagent Amount
pSB1C3 33 uL
DW 2 uL
0.1% BSA 5 uL
10x M Buffer 5 uL
EcoR I 2 uL
Pst I 3 uL
Total 50 uL

→37℃, 60 min
→10 uLの6x Sample bufferを加え，次のように泳動

電気泳動＆ゲル抽

レーン２～５は濃度測定のため各1 uLのDNAを泳動した．

HokkaidoU Japan 20100902a.jpg
Lane DNA
1 λ/Hind III, EcoR I
2 50 base ladder
3 Heat shock promotor
4 RBS
5 RFP protein coding
6 double Terminator
7 blank
8 RFP reporter (1-5A) 15 uL each
9
10
11
12 blank
13 pSB1C3 15 uL each
14
15
16
17 blank

それぞれゲルから切り出し(210 mg，220 mg)，抽出キットで20 uLにした．
この時点でRFP 104 ng/uL，pSB1C3 33 ng/uL．

Ligation

Reagent Amount
RFP 1.5 uL
pSB1C3 1.5 uL
ligation Kit I 3 uL
T4 ligase 0.5 uL
Total 6.5 uL
Reagent Amount
RFP 1.5 uL
pSB1C3 1.5 uL
Mighty Mix 3 uL
T4 ligase 0.5 uL
Total 6.5 uL
Reagent Amount
RFP 0.8 uL
pUC119 4 uL
ligation Kit I 4.8 uL
T4 ligase 0.5 uL
Total 10.5 uL
Reagent Amount
RFP 0.8 uL
pUC119 4 uL
Mighty Mix 4.8 uL
T4 ligase 0.5 uL
Total 10.5 uL

→16℃, 30 min
→Transformation

=HSP, RBS, RFP, dTのゲル抽=
Retrieved from "http://2010.igem.org/Team:HokkaidoU_Japan/Notebook/September2"