Team:HokkaidoU Japan/Notebook/September2

From 2010.igem.org

(Difference between revisions)
(Ligation)
(RFPとpSB1C3,RFPとpUC119のDigestion/ Ligation/ Transformation)
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=RFPとpSB1C3,RFPとpUC119のDigestion/ Ligation/ Transformation=
+
=Digestion, Ligation and Transformation of pUC119, RFP, pSB1C3 and RFP=
 +
 
{|style="text-align:center; class="protocol"
{|style="text-align:center; class="protocol"
|-
|-
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|35 uL
|35 uL
|-
|-
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|pUC119(cut済み)
+
|pUC119(pre cut)
|10 ng/uL
|10 ng/uL
|3000 bp
|3000 bp
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==Digestion==
==Digestion==
 +
{|style="text-align:center; float: left;" class="protocol"
{|style="text-align:center; float: left;" class="protocol"
!Reagent
!Reagent
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|}
|}
<div style="clear:both;"></div>
<div style="clear:both;"></div>
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→37℃, 60 min<br>
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→Incubated at 37C for 60 min<br>
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→10 uLの6x Sample bufferを加え,次のように泳動
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→Adde 10 uL 6x Sample buffer and electrophoresed
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==電気泳動&ゲル抽==
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==Electrophoresis and gel extraction==
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レーン2~5は濃度測定のため各1 uLのDNAを泳動した.
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To check concentration lanes 2 through 5 contained 1uL of DNA solution
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[[Image:HokkaidoU Japan 20100902a.jpg|200px|right|thumb|]]
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[[Image:HokkaidoU Japan 20100902a.jpg|200px|right|thumb|Electrophoresis of digestion product]]
{|class="protocol"
{|class="protocol"
|'''Lane'''
|'''Lane'''
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|-
|-
|1
|1
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|λ/''Hin''d III, EcoR I
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|[http://2010.igem.org/Image:HokkaidoU_Pictures_DNA_Marker.png Lambda/''Hin''d III, EcoR I]
|-
|-
|2
|2
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|blank
|blank
|}
|}
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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.<br>
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Bands were exised (210 mg and 220 mg acordingly)
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この時点でRFP 104 ng/uL,pSB1C3 33 ng/uL.
+
Final volume was made to be 20 uL, with disregard to promega`s protocol<br>
 +
At this point concentrations were: RFP 104 ng/uL,pSB1C3 33 ng/uL.
==Ligation==
==Ligation==
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|}
|}
<div style="clear:both;"></div>
<div style="clear:both;"></div>
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→16℃, 30 min<br>
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→Incubated 16C for min<br>
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→Transformation
+
→Transformed
=HSP, RBS, RFP, dTのゲル抽=
=HSP, RBS, RFP, dTのゲル抽=

Revision as of 17:21, 27 September 2010

Digestion, Ligation and Transformation of pUC119, RFP, pSB1C3 and RFP

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

→Incubated at 37C for 60 min
→Adde 10 uL 6x Sample buffer and electrophoresed

Electrophoresis and gel extraction

To check concentration lanes 2 through 5 contained 1uL of DNA solution

Electrophoresis of digestion product
Lane DNA
1 Lambda/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

Bands were exised (210 mg and 220 mg acordingly) Final volume was made to be 20 uL, with disregard to promega`s protocol
At this point concentrations were: 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

→Incubated 16C for min
→Transformed

=HSP, RBS, RFP, dTのゲル抽=