Team:HokkaidoU Japan/Notebook/September2

From 2010.igem.org

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{{Template:HokkaidoU_Japan}}<div class="linkbar"><div class="prev">[[Team:HokkaidoU_Japan/Notebook/September1|September 1]]</div>[[Team:HokkaidoU_Japan/Notebook|Notebook]]<div class="next">[[Team:HokkaidoU_Japan/Notebook/September3|September 3]]</div></div>
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=[[Team:HokkaidoU_Japan/Protocols|Transformation]] of pUC119, RFP, pSB1C3 and RFP=
 +
 
 +
==Parts information==
 +
{|style="text-align:center; class="protocol"
 +
|-
 +
!DNA
 +
!Concentration
 +
!Length
 +
!Amount
 +
|-
 +
|RFP ([[Team:HokkaidoU_Japan/Parts#BioBricks|1-5A]])
 +
|80 ng/uL
 +
|1000 bp
 +
|26 uL
 +
|-
 +
|pSB1C3
 +
|20 ng/uL
 +
|2000 bp
 +
|35 uL
 +
|-
 +
|pUC119(pre cut)
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|10 ng/uL
 +
|3000 bp
 +
|28 uL
 +
|}
 +
 
 +
==Digestion==
 +
 
 +
{|style="text-align:center; float: left;" class="protocol"
 +
!Reagent
 +
!Amount
 +
|-
 +
|RFP ([[Team:HokkaidoU_Japan/Parts#BioBricks|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
 +
|-
 +
|style="border-top:1px solid #996;"|'''Total'''
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|style="border-top:1px solid #996;"|'''50 uL'''
 +
|}
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 +
{|style="text-align:center; float: left;" class="protocol"
 +
!Reagent
 +
!Amount
 +
|-
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|pSB1C3
 +
|33 uL
 +
|-
 +
|DW
 +
|2 uL
 +
|-
 +
|0.1% BSA
 +
|5 uL
 +
|-
 +
|10x M Buffer
 +
|5 uL
 +
|-
 +
|EcoR I
 +
|2 uL
 +
|-
 +
|Pst I
 +
|3 uL
 +
|-
 +
|style="border-top:1px solid #996;"|'''Total'''
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|style="border-top:1px solid #996;"|'''50 uL'''
 +
|}
 +
<div style="clear:both;"></div>
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→Incubated at 37C for 60 min<br>
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→Adde 10 uL 6x Sample buffer and electrophoresed
 +
 
 +
==Electrophoresis and [[Team:HokkaidoU_Japan/Protocols|gel extraction]]==
 +
To check concentration lanes 2 through 5 contained 1uL of DNA solution
 +
[[Image:HokkaidoU Japan 20100902a.jpg|200px|right|thumb|Electrophoresis of digestion product]]
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{|class="protocol"
 +
|'''Lane'''
 +
|'''DNA'''
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|-
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|1
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|[http://2010.igem.org/Image:HokkaidoU_Pictures_DNA_Marker.png λ/''Hin''d III, EcoR I]
 +
|-
 +
|2
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|50 base ladder
 +
|-
 +
|3
 +
|Heat shock promotor
 +
|-
 +
|4
 +
|RBS
 +
|-
 +
|5
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|RFP protein coding
 +
|-
 +
|6
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|double Terminator
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|-
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|7
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|blank
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|-
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|8
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|style="border-left:1px solid #000;" rowspan="4"|RFP reporter ([[Team:HokkaidoU_Japan/Parts#BioBricks|1-5A]]) 15 uL each
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|-
 +
|9
 +
|-
 +
|10
 +
|-
 +
|11
 +
|-
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|12
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|blank
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|-
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|13
 +
|rowspan="4" style="border-left:1px solid #000;"|pSB1C3 15 uL each
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|-
 +
|14
 +
|-
 +
|15
 +
|-
 +
|16
 +
|-
 +
|17
 +
|blank
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|}
 +
Bands were exised (210 mg and 220 mg acordingly)
 +
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==
 +
{|style="text-align:center;float: left;" class="protocol"
 +
!Reagent
 +
!Amount
 +
|-
 +
|RFP
 +
|1.5 uL
 +
|-
 +
|pSB1C3
 +
|1.5 uL
 +
|-
 +
|ligation Kit I
 +
|3 uL
 +
|-
 +
|T4 ligase
 +
|0.5 uL
 +
|-
 +
|style="border-top:1px solid #996;"|'''Total'''
 +
|style="border-top:1px solid #996;"|'''6.5 uL'''
 +
|}
 +
 
 +
{|style="text-align:center; float: left;" class="protocol"
 +
!Reagent
 +
!Amount
 +
|-
 +
|RFP
 +
|1.5 uL
 +
|-
 +
|pSB1C3
 +
|1.5 uL
 +
|-
 +
|Mighty Mix
 +
|3 uL
 +
|-
 +
|T4 ligase
 +
|0.5 uL
 +
|-
 +
|style="border-top:1px solid #996;"|'''Total'''
 +
|style="border-top:1px solid #996;"|'''6.5 uL'''
 +
|}
 +
 
 +
{|style="text-align:center; float: left;" class="protocol"
 +
!Reagent
 +
!Amount
 +
|-
 +
|RFP
 +
|0.8 uL
 +
|-
 +
|pUC119
 +
|4 uL
 +
|-
 +
|ligation Kit I
 +
|4.8 uL
 +
|-
 +
|T4 ligase
 +
|0.5 uL
 +
|-
 +
|style="border-top:1px solid #996;"|'''Total'''
 +
|style="border-top:1px solid #996;"|'''10.5 uL'''
 +
|}
 +
 
 +
{|style="text-align:center; float: left;" class="protocol"
 +
!Reagent
 +
!Amount
 +
|-
 +
|RFP
 +
|0.8 uL
 +
|-
 +
|pUC119
 +
|4 uL
 +
|-
 +
|Mighty Mix
 +
|4.8 uL
 +
|-
 +
|T4 ligase
 +
|0.5 uL
 +
|-
 +
|style="border-top:1px solid #996;"|'''Total'''
 +
|style="border-top:1px solid #996;"|'''10.5 uL'''
 +
|}
 +
<div style="clear:both;"></div>
 +
→Incubated 16C for min<br>
 +
→[[Team:HokkaidoU_Japan/Protocols|Transformation]]

Latest revision as of 08:02, 27 October 2010

Transformation of pUC119, RFP, pSB1C3 and RFP

Parts information

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 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

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

Transformation