Template:Kyoto/Notebook/Aug 17

From 2013.igem.org

(Difference between revisions)
(→Aug 17)
(→Colony PCR)
 
(21 intermediate revisions not shown)
Line 9: Line 9:
|8/15 BBa_K112806(lysis2) -(1)||828
|8/15 BBa_K112806(lysis2) -(1)||828
|-
|-
-
|negative control||--
+
|NC||--
|}
|}
{| class="wikitable"
{| class="wikitable"
Line 18: Line 18:
|5min||30s||30s||1min||30cycles
|5min||30s||30s||1min||30cycles
|}
|}
-
<!-- [[File:image]] -->
 
{| class="wikitable"
{| class="wikitable"
!Sample||base pair
!Sample||base pair
Line 24: Line 23:
|8/10 BBa_Q0400 -(1)||3995
|8/10 BBa_Q0400 -(1)||3995
|-
|-
-
|negative control||--
+
|NC||--
|}
|}
{| class="wikitable"
{| class="wikitable"
Line 33: Line 32:
|5min||30s||30s||30s||30cycles
|5min||30s||30s||30s||30cycles
|}
|}
-
[[File:igku_Aug19electrophoresis2.png]]
 
</div>
</div>
-
<br>
+
 
 +
===Electrophoresis===
 +
<div class="experiment">
 +
<span class="author">No name</span>
 +
{| class="wikitable"
 +
!Lane||Sample
 +
|-
 +
|1||1kbp ladder
 +
|-
 +
|2||K112806(lysis2) -1
 +
|-
 +
|3||NC
 +
|-
 +
|4||Q04400(tetR) -1
 +
|-
 +
|5||NC
 +
|-
 +
|6||1kbp ladder
 +
|}
 +
[[File:Igku Aug17colonyPCR.jpg]]<br>
 +
</div>
 +
 
 +
===Liquid Culture===
 +
<div class="experiment">
 +
<span class="author">Nakamoto</span>
 +
{| class="wikitable"
 +
!Sample||medium
 +
|-
 +
|8/15 BBa_K112806(lysis2) -(1)||Plusgrow medium(+CP)
 +
|-
 +
|8/15 BBa_Q04400(tetR) -(1)||Plusgrow medium(+Amp)
 +
|-
 +
|8/16 pBR322 -(1)||Plusgrow medium(+Amp)
 +
|}
 +
</div>
 +
 
 +
===Master Plate===
 +
<div class="experiment">
 +
<span class="author">Nakamoto</span>
 +
{| class="wikitable"
 +
!Number||Use LB plate (+CP)
 +
|-
 +
|1||BBa_K112806(lysis2) -(1)
 +
|}
 +
</div>
 +
<div class="experiment">
 +
{| class="wikitable"
 +
!Number||Use LB plate (+Kana)
 +
|-
 +
|1||BBa_Q04400(tetR) -(1)
 +
|}
 +
</div>
 +
===Miniprep===
 +
<div class="experiment">
 +
<span class="author">Nakamoto and Murakami</span>
 +
{|class="wikitable"
 +
!DNA||concentration[&micro;g/mL]||260/280||260/230
 +
|-
 +
|K748002(lysis3) -(1)||330||1.66||2.08
 +
|-
 +
|B0015(DT) -(1)||188||1.73||1.82
 +
|-
 +
|R0061(Plux) -(1)||76||1.64||1.30
 +
|-
 +
|K117000(lysis1) -(1)||286||1.71||2.01
 +
|-
 +
|K117000(lysis1) -(2)||248||1.69||1.78
 +
|-
 +
|I732020(RBS-lacZ&alpha;-DT) -(1)||198||1.71||1.93
 +
|-
 +
|I13504(RBS-GFP-DT) -(1)||248||1.69||2.03
 +
|-
 +
|I13504(RBS-GFP-DT) -(2)||246||1.55||1.73
 +
|-
 +
|I0462(RBS-luxR-DT) -(1)||326||1.65||1.74
 +
|}
 +
</div>
 +
 
 +
===Restriction Enzyme Digestion===
 +
<div class="experiment">
 +
<span class="author">Murata, Murakami, Nakamoto, Ashida, Matsumoto and Yoshida</span>
 +
{| class="wikitable"
 +
! ||8/11 J23100 -(2)||SpeI||PstI||Buffer||MilliQ||total
 +
|-
 +
|2 cuts||4.0&micro;L||1.0&micro;L||1.0&micro;L||3.0&micro;L||21&micro;L||30&micro;L
 +
|-
 +
|1 cut||1.3&micro;L||0.2&micro;L||--||1.0&micro;L||7.5&micro;L||10&micro;L
 +
|-
 +
|1 cut||1.3&micro;L||--||0.2&micro;L||1.0&micro;L||7.5&micro;L||10&micro;L
 +
|-
 +
|NC||1.3&micro;L||--||--||1.0&micro;L||7.7&micro;L||10&micro;L
 +
|}
 +
{| class="wikitable"
 +
! ||8/15 B0034(RBS) -(2)||SpeI||PstI||Buffer||MilliQ||total
 +
|-
 +
|2 cuts||6.0&micro;L||1.0&micro;L||1.0&micro;L||3.0&micro;L||19&micro;L||30&micro;L
 +
|-
 +
|1 cut||2.0&micro;L||0.2&micro;L||--||1.0&micro;L||6.8&micro;L||10&micro;L
 +
|-
 +
|1 cut||2.0&micro;L||--||0.2&micro;L||1.0&micro;L||6.8&micro;L||10&micro;L
 +
|-
 +
|NC||2.0&micro;L||--||--||1.0&micro;L||7.0&micro;L||10&micro;L
 +
|}
 +
{| class="wikitable"
 +
! ||8/17 K117000(lysis1) -(1)||XbaI||PstI||BSA||Buffer||MilliQ||total
 +
|-
 +
|2 cuts||2.0&micro;L||1.0&micro;L||1.0&micro;L||3.0&micro;L||3.0&micro;L||20&micro;L||30&micro;L
 +
|-
 +
|1 cut||1.0&micro;L||0.2&micro;L||--||1.0&micro;L||1.0&micro;L||6.8&micro;L||10&micro;L
 +
|-
 +
|1 cut||1.0&micro;L||--||0.2&micro;L||1.0&micro;L||1.0&micro;L||6.8&micro;L||10&micro;L
 +
|-
 +
|NC||1.0&micro;L||--||--||1.0&micro;L||1.0&micro;L||7.0&micro;L||10&micro;L
 +
|}
 +
{| class="wikitable"
 +
! ||8/17 K748002(lysis3) -(1)||XbaI||PstI||BSA||Buffer||MilliQ||total
 +
|-
 +
|2 cuts||2.0&micro;L||1.0&micro;L||1.0&micro;L||3.0&micro;L||3.0&micro;L||20&micro;L||30&micro;L
 +
|-
 +
|1 cut||1.0&micro;L||0.2&micro;L||--||1.0&micro;L||1.0&micro;L||6.8&micro;L||10&micro;L
 +
|-
 +
|1 cut||1.0&micro;L||--||0.2&micro;L||1.0&micro;L||1.0&micro;L||6.8&micro;L||10&micro;L
 +
|-
 +
|NC||1.0&micro;L||--||--||1.0&micro;L||1.0&micro;L||7.0&micro;L||10&micro;L
 +
|}
 +
{| class="wikitable"
 +
! ||8/17 I13504(RBS-GFP-DT) -(1)||XbaI||PstI||BSA||Buffer||MilliQ||total
 +
|-
 +
|2 cuts||2.0&micro;L||1.0&micro;L||1.0&micro;L||3.0&micro;L||3.0&micro;L||20&micro;L||30&micro;L
 +
|-
 +
|1 cut||1.0&micro;L||0.2&micro;L||--||1.0&micro;L||1.0&micro;L||6.8&micro;L||10&micro;L
 +
|-
 +
|1 cut||1.0&micro;L||--||0.2&micro;L||1.0&micro;L||1.0&micro;L||6.8&micro;L||10&micro;L
 +
|-
 +
|NC||1.0&micro;L||--||--||1.0&micro;L||1.0&micro;L||7.0&micro;L||10&micro;L
 +
|}
 +
{| class="wikitable"
 +
! ||8/17 I0462(RBS-luxR-DT) -(1)||XbaI||PstI||BSA||Buffer||MilliQ||total
 +
|-
 +
|2 cuts||2.0&micro;L||1.0&micro;L||1.0&micro;L||3.0&micro;L||3.0&micro;L||20&micro;L||30&micro;L
 +
|-
 +
|1 cut||1.0&micro;L||0.2&micro;L||--||1.0&micro;L||1.0&micro;L||6.8&micro;L||10&micro;L
 +
|-
 +
|1 cut||1.0&micro;L||--||0.2&micro;L||1.0&micro;L||1.0&micro;L||6.8&micro;L||10&micro;L
 +
|-
 +
|NC||1.0&micro;L||--||--||1.0&micro;L||1.0&micro;L||7.0&micro;L||10&micro;L
 +
|}
 +
{| class="wikitable"
 +
! ||8/17 I732020(RBS-lacZ&alpha;-DT) -(1)||XbaI||PstI||BSA||Buffer||MilliQ||total
 +
|-
 +
|2 cuts||2.0&micro;L||1.0&micro;L||1.0&micro;L||3.0&micro;L||3.0&micro;L||20&micro;L||30&micro;L
 +
|-
 +
|1 cut||1.0&micro;L||0.2&micro;L||--||1.0&micro;L||1.0&micro;L||6.8&micro;L||10&micro;L
 +
|-
 +
|1 cut||1.0&micro;L||--||0.2&micro;L||1.0&micro;L||1.0&micro;L||6.8&micro;L||10&micro;L
 +
|-
 +
|NC||1.0&micro;L||--||--||1.0&micro;L||1.0&micro;L||7.0&micro;L||10&micro;L
 +
|}
 +
{| class="wikitable"
 +
! ||8/11 R6061 -(1)||SpeI||PstI||Buffer||MilliQ||total
 +
|-
 +
|2 cuts||8.0&micro;L||1.0&micro;L||1.0&micro;L||3.0&micro;L||17&micro;L||30&micro;L
 +
|-
 +
|1 cut||3.0&micro;L||0.2&micro;L||--||1.0&micro;L||5.8&micro;L||10&micro;L
 +
|-
 +
|1 cut||3.0&micro;L||--||0.2&micro;L||1.0&micro;L||5.8&micro;L||10&micro;L
 +
|-
 +
|NC||3.0&micro;L||--||--||1.0&micro;L||6.0&micro;L||10&micro;L
 +
|}
 +
*incubate at 37 &deg;C, for 1 h
 +
</div>
 +
 
 +
===Electrophoresis===
 +
<div class="experiment">
 +
<span class="author">No name</span>
 +
{| class="wikitable"
 +
!Lane||Sample||Enzyme1||Enzyme2
 +
|-
 +
|1||J23100 -&#9313;||SpeI||PstI
 +
|-
 +
|2||J23100 -&#9313;||SpeI||--
 +
|-
 +
|3||J23100 -&#9313;||--||PstI
 +
|-
 +
|4||J23100 -&#9313;||--||--
 +
|-
 +
|5||B0034(RBS) -(2)||SpeI||PstI
 +
|-
 +
|6||B0034(RBS) -(2)||SpeI||--
 +
|-
 +
|7||B0034(RBS) -(2)||--||PstI
 +
|-
 +
|8||B0034(RBS) -(2)||--||--
 +
|-
 +
|9||1kb ladder||--||--
 +
|-
 +
|10||K11700(lysis1) -(1)||XbaI||PstI
 +
|-
 +
|11||K11700(lysis1) -(1)||XbaI||--
 +
|-
 +
|12||K11700(lysis1) -(1)||--||PstI
 +
|-
 +
|13||K11700(lysis1) -(1)||--||--
 +
|-
 +
|14||K748002(lysis3) -(1)||XbaI||PstI
 +
|-
 +
|15||K748002(lysis3) -(1)||XbaI||--
 +
|-
 +
|16||K748002(lysis3) -(1)||--||PstI
 +
|-
 +
|17||K748002(lysis3) -(1)||--||--
 +
|}
 +
[[File:Igku Aug17 Electrophoresis(1).jpg]]<br>
 +
{| class="wikitable"
 +
!Lane||Sample||Enzyme1||Enzyme2
 +
|-
 +
|18||I13504(RBS-GFP-DT) -(1)||XbaI||PstI
 +
|-
 +
|19||I13504(RBS-GFP-DT) -(1)||XbaI||--
 +
|-
 +
|20||I13504(RBS-GFP-DT) -(1)||--||PstI
 +
|-
 +
|21||I13504(RBS-GFP-DT) -(1)||--||--
 +
|-
 +
|22||I0462(RBS-luxR-DT) -(1)||XbaI||PstI
 +
|-
 +
|23||I0462(RBS-luxR-DT) -(1)||XbaI||--
 +
|-
 +
|24||I0462(RBS-luxR-DT) -(1)||--||PstI
 +
|-
 +
|25||I0462(RBS-luxR-DT) -(1)||--||--
 +
|-
 +
|26||1kb ladder||--||--
 +
|-
 +
|27||I732020(RBS-lacZ&alpha;-DT) -(1)||XbaI||PstI
 +
|-
 +
|28||I732020(RBS-lacZ&alpha;-DT) -(1)||XbaI||--
 +
|-
 +
|29||I732020(RBS-lacZ&alpha;-DT) -(1)||--||PstI
 +
|-
 +
|30||I732020(RBS-lacZ&alpha;-DT) -(1)||--||--
 +
|-
 +
|31||R0061(Plux) -(1)||SpeI||PstI
 +
|-
 +
|32||R0061(Plux) -(1)||SpeI||--
 +
|-
 +
|33||R0061(Plux) -(1)||--||PstI
 +
|-
 +
|34||R0061(Plux) -(1)||--||--
 +
|}
 +
[[File:Igku Aug17 Electrophoresis(2).jpg]]<br>
 +
</div>
 +
 
 +
===Miniprep===
 +
<div class="experiment">
 +
<span class="author">Murata</span>
 +
{|class="wikitable"
 +
!DNA||concentration[&micro;g/mL]||260/280||260/230
 +
|-
 +
|8/17 PBR322(TaKaRa) -(1)||47||1.68||1.55
 +
|-
 +
|8/17 K112806(lysis2) -(1)||64||1.67||1.70
 +
|}
 +
</div>

Latest revision as of 07:41, 27 September 2013

Contents

Aug 17

Colony PCR

Nakamoto

Samplebase pair
8/15 BBa_K112806(lysis2) -(1)828
NC--
PreDenatureDenatureAnnealingExtensioncycle
94°C94°C55°C68°C--
5min30s30s1min30cycles
Samplebase pair
8/10 BBa_Q0400 -(1)3995
NC--
PreDenatureDenatureAnnealingExtensioncycle
94°C94°C55°C68°C--
5min30s30s30s30cycles

Electrophoresis

No name

LaneSample
11kbp ladder
2K112806(lysis2) -1
3NC
4Q04400(tetR) -1
5NC
61kbp ladder

Igku Aug17colonyPCR.jpg

Liquid Culture

Nakamoto

Samplemedium
8/15 BBa_K112806(lysis2) -(1)Plusgrow medium(+CP)
8/15 BBa_Q04400(tetR) -(1)Plusgrow medium(+Amp)
8/16 pBR322 -(1)Plusgrow medium(+Amp)

Master Plate

Nakamoto

NumberUse LB plate (+CP)
1BBa_K112806(lysis2) -(1)
NumberUse LB plate (+Kana)
1BBa_Q04400(tetR) -(1)

Miniprep

Nakamoto and Murakami

DNAconcentration[µg/mL]260/280260/230
K748002(lysis3) -(1)3301.662.08
B0015(DT) -(1)1881.731.82
R0061(Plux) -(1)761.641.30
K117000(lysis1) -(1)2861.712.01
K117000(lysis1) -(2)2481.691.78
I732020(RBS-lacZα-DT) -(1)1981.711.93
I13504(RBS-GFP-DT) -(1)2481.692.03
I13504(RBS-GFP-DT) -(2)2461.551.73
I0462(RBS-luxR-DT) -(1)3261.651.74

Restriction Enzyme Digestion

Murata, Murakami, Nakamoto, Ashida, Matsumoto and Yoshida

8/11 J23100 -(2)SpeIPstIBufferMilliQtotal
2 cuts4.0µL1.0µL1.0µL3.0µL21µL30µL
1 cut1.3µL0.2µL--1.0µL7.5µL10µL
1 cut1.3µL--0.2µL1.0µL7.5µL10µL
NC1.3µL----1.0µL7.7µL10µL
8/15 B0034(RBS) -(2)SpeIPstIBufferMilliQtotal
2 cuts6.0µL1.0µL1.0µL3.0µL19µL30µL
1 cut2.0µL0.2µL--1.0µL6.8µL10µL
1 cut2.0µL--0.2µL1.0µL6.8µL10µL
NC2.0µL----1.0µL7.0µL10µL
8/17 K117000(lysis1) -(1)XbaIPstIBSABufferMilliQtotal
2 cuts2.0µL1.0µL1.0µL3.0µL3.0µL20µL30µL
1 cut1.0µL0.2µL--1.0µL1.0µL6.8µL10µL
1 cut1.0µL--0.2µL1.0µL1.0µL6.8µL10µL
NC1.0µL----1.0µL1.0µL7.0µL10µL
8/17 K748002(lysis3) -(1)XbaIPstIBSABufferMilliQtotal
2 cuts2.0µL1.0µL1.0µL3.0µL3.0µL20µL30µL
1 cut1.0µL0.2µL--1.0µL1.0µL6.8µL10µL
1 cut1.0µL--0.2µL1.0µL1.0µL6.8µL10µL
NC1.0µL----1.0µL1.0µL7.0µL10µL
8/17 I13504(RBS-GFP-DT) -(1)XbaIPstIBSABufferMilliQtotal
2 cuts2.0µL1.0µL1.0µL3.0µL3.0µL20µL30µL
1 cut1.0µL0.2µL--1.0µL1.0µL6.8µL10µL
1 cut1.0µL--0.2µL1.0µL1.0µL6.8µL10µL
NC1.0µL----1.0µL1.0µL7.0µL10µL
8/17 I0462(RBS-luxR-DT) -(1)XbaIPstIBSABufferMilliQtotal
2 cuts2.0µL1.0µL1.0µL3.0µL3.0µL20µL30µL
1 cut1.0µL0.2µL--1.0µL1.0µL6.8µL10µL
1 cut1.0µL--0.2µL1.0µL1.0µL6.8µL10µL
NC1.0µL----1.0µL1.0µL7.0µL10µL
8/17 I732020(RBS-lacZα-DT) -(1)XbaIPstIBSABufferMilliQtotal
2 cuts2.0µL1.0µL1.0µL3.0µL3.0µL20µL30µL
1 cut1.0µL0.2µL--1.0µL1.0µL6.8µL10µL
1 cut1.0µL--0.2µL1.0µL1.0µL6.8µL10µL
NC1.0µL----1.0µL1.0µL7.0µL10µL
8/11 R6061 -(1)SpeIPstIBufferMilliQtotal
2 cuts8.0µL1.0µL1.0µL3.0µL17µL30µL
1 cut3.0µL0.2µL--1.0µL5.8µL10µL
1 cut3.0µL--0.2µL1.0µL5.8µL10µL
NC3.0µL----1.0µL6.0µL10µL
  • incubate at 37 °C, for 1 h

Electrophoresis

No name

LaneSampleEnzyme1Enzyme2
1J23100 -②SpeIPstI
2J23100 -②SpeI--
3J23100 -②--PstI
4J23100 -②----
5B0034(RBS) -(2)SpeIPstI
6B0034(RBS) -(2)SpeI--
7B0034(RBS) -(2)--PstI
8B0034(RBS) -(2)----
91kb ladder----
10K11700(lysis1) -(1)XbaIPstI
11K11700(lysis1) -(1)XbaI--
12K11700(lysis1) -(1)--PstI
13K11700(lysis1) -(1)----
14K748002(lysis3) -(1)XbaIPstI
15K748002(lysis3) -(1)XbaI--
16K748002(lysis3) -(1)--PstI
17K748002(lysis3) -(1)----

Igku Aug17 Electrophoresis(1).jpg

LaneSampleEnzyme1Enzyme2
18I13504(RBS-GFP-DT) -(1)XbaIPstI
19I13504(RBS-GFP-DT) -(1)XbaI--
20I13504(RBS-GFP-DT) -(1)--PstI
21I13504(RBS-GFP-DT) -(1)----
22I0462(RBS-luxR-DT) -(1)XbaIPstI
23I0462(RBS-luxR-DT) -(1)XbaI--
24I0462(RBS-luxR-DT) -(1)--PstI
25I0462(RBS-luxR-DT) -(1)----
261kb ladder----
27I732020(RBS-lacZα-DT) -(1)XbaIPstI
28I732020(RBS-lacZα-DT) -(1)XbaI--
29I732020(RBS-lacZα-DT) -(1)--PstI
30I732020(RBS-lacZα-DT) -(1)----
31R0061(Plux) -(1)SpeIPstI
32R0061(Plux) -(1)SpeI--
33R0061(Plux) -(1)--PstI
34R0061(Plux) -(1)----

Igku Aug17 Electrophoresis(2).jpg

Miniprep

Murata

DNAconcentration[µg/mL]260/280260/230
8/17 PBR322(TaKaRa) -(1)471.681.55
8/17 K112806(lysis2) -(1)641.671.70
Retrieved from "http://2013.igem.org/Template:Kyoto/Notebook/Aug_17"