Team:Kyoto/Notebook/DMS Synthesis

From 2014.igem.org

(Difference between revisions)
Line 12: Line 12:
<h1>アメフラシノート</h1>
<h1>アメフラシノート</h1>
-
//・Liquid Culture//
+
 
<a name=“半角小文字日付（ex. September23）” class=“kyoto-jump”>
<a name=“半角小文字日付（ex. September23）” class=“kyoto-jump”>
<h3>日付</h3>
<h3>日付</h3>
Line 22: Line 22:
</table>
</table>
-
//・Gel Extraction//
+
 
<a name=“半角小文字日付（ex. September23）” class=“kyoto-jump”>
<a name=“半角小文字日付（ex. September23）” class=“kyoto-jump”>
<h3>日付</h3>
<h3>日付</h3>
Line 32: Line 32:
</table>
</table>
-
//・Electrophoresis//
+
 
<a name=“半角小文字日付（ex. September23）” class=“kyoto-jump”>
<a name=“半角小文字日付（ex. September23）” class=“kyoto-jump”>
<h3>日付</h3>
<h3>日付</h3>
Line 42: Line 42:
</table>
</table>
-
//・Miniprep/DNA Purification//
+
 
<a name=“半角小文字日付（ex. September23）” class=“kyoto-jump”>
<a name=“半角小文字日付（ex. September23）” class=“kyoto-jump”>
<h3>日付</h3>
<h3>日付</h3>
Line 52: Line 52:
</table>
</table>
-
//・Restriction Enzyme Digestion//
+
 
<a name=“半角小文字日付（ex. September23）” class=“kyoto-jump”>
<a name=“半角小文字日付（ex. September23）” class=“kyoto-jump”>
<h3>日付</h3>
<h3>日付</h3>
Line 62: Line 62:
</table>
</table>
-
//・Ligation//
+
 
<a name=“半角小文字日付（ex. September23）” class=“kyoto-jump”>
<a name=“半角小文字日付（ex. September23）” class=“kyoto-jump”>
<h3>日付</h3>
<h3>日付</h3>
Line 72: Line 72:
</table>
</table>
-
//・Transformation//
+
 
<a name=“半角小文字日付（ex. September23）” class=“kyoto-jump”>
<a name=“半角小文字日付（ex. September23）” class=“kyoto-jump”>
<h3>日付</h3>
<h3>日付</h3>
Line 82: Line 82:
</table>
</table>
-
//・Dephosphorylation//
+
 
<a name=“半角小文字日付（ex. September23）” class=“kyoto-jump”>
<a name=“半角小文字日付（ex. September23）” class=“kyoto-jump”>
<h3>日付</h3>
<h3>日付</h3>

Revision as of 07:23, 23 September 2014

count down

edit

アメフラシノート

日付

Liquid Culture

名前
SampleMedium
サンプル名培地

日付

Gel Extraction

名前
LaneSample
レーン番号サンプル名

日付

Electrophoresis

名前
LaneSampleEnsyme1Ensyme2
レーン番号サンプル名制限酵素1制限酵素2

日付

Miniprep/DNA Purification

名前
SampleConcentration(µg/mL)260/280260/230
サンプル名(Concentration)(260/280)(260/280)

日付

Restriction Enzyme Digestion

名前
SampleEcoR1Xba1Spe1Pst1BufferBSAMilliQTotal
サンプル名(サンプル名)(E)(X)(S)(P)(HorM)(Buffer)(BSA)(MilliQ)(Total)

日付

Ligation

名前
VectorInsertBufferLigaseMilliQTotal
ベクター名(ベクター)インサート名(インサート)(HorM)(Buffer)Ligase名(Ligase)(MilliQ)(Total)

日付

Transformation

名前
SampleCompetentCellsTotalMedium
サンプル名(サンプル)コンピ名(コンピ)(Total)培地

日付

Dephosphorylation

名前
Sample
サンプル名(サンプル)

7/31

Restriction Enzyme Digestion

Nakamura Matsumoto
SampleVolumeEcoR1Spe1Buffer(H)MilliQTOTAL
3/24 PCR(dddD)cloning3.211321.830
pSB1C318.51136.530
pSB1C3(control)6.20002.810

8/1

Gel Extraction

Yasuda Murata
LaneDNA
11kbp ladder
23/24 PCR(dddD)cloning E,S
33/24 PCR(dddD)cloning E,S
4Blank
5pSB1C3
6pSB1C3
7Blank
81kbp ladder
93/24 PCR(dddD)cloning(control)
10pSB1C3(control)

8/1

Ligation

Yasuda
Vectorinserter10xt4LigaseBuffert4LigaseMilliQTOTAL
1.5 µL(8/1 pSB1C3 2047bp 13.3µg/mL 20μg) 6.9 µL(8/1 dddD 2545bp 10.8µg/mL 74.60µg) 2µL 1µL 8.6µL 20µL

8/1

transformation

Murata
NameSample/(µl)Competent Cells/(µl)Total/(µl)Plate
pSB1C3330165SOC(132µl)

8/2

PCR Cloning

Murata
dddD 121µg/µL
2x KAPA HiFiHotStartReadyMix12.5µL
F Pre-dddD cloning 620.75µL
Rv Spe1-dddDcloning 680.75µL
MilliQ11µL
TOTAL25µL

8/2

Colony PCR

Honda Matsumoto Nakamura
Master Mix
Quick Taq15µL
VF20.6µL
VR0.6µL
MilliQ13.8µL
TOTAL30µL

8/2

Restriction Enzyme Digestion

Nakamura Matsumoto
SampleVolumeEcoR1Spe1BufferMilliQTOTAL
8/2 dddD cloning8.211316.830
8/2 dddD cloning control0.410018.610
pSB1C3(control)6.2003(H)2.810

8/3

Electrophoresis for confirmation

Murata
LaneSample
1pSB1C3 dddD 1
2pSB1C3 dddD 2
3pSB1C3 dddD 3
41kb ladder(3µL)
51kb ladder(1µL)

8/3

Gel Extraction

Tatsui
LaneDNA
11kbp ladder
2Blank
3pSB1C3 25µμL 2070bp
4Blank
5pSB1C3 5µL
6Blank
78/2 cloning dddD 25µL 2544bp
8Blank
93/24 PCR(dddD)cloning(control)
108/2 cloning dddD 5µL

9/14

Gel Extraction

Honda,Yasuda
LaneDNA
11kbp ladder
23/24 PCR(dddD)cloning E,S
39/13SAMmT(control)
49/13SAMmT(EP)
59/13Pcon-pSB1C3(control)
69/13Pcon-pSB1C3(XS)

9/13

Column cleanup

Yasuda
Sampledensity(µg/mL)260/280260/230
9/12 AT-EP2.31.331.11
9/12 AT-His-XP2.01.330.86

9/13

Ligation

Honda
sumpleVectorInsertLigase2xBufferMilliQ
9/13 column cleanup AT(EP)+9/8 pSB1C3(EP)13150
9/13 column cleanup AT-His(XP)+9/13 pSB1C3(XP)13150

9/13

Miniprep

Honda
Sampledensity(µg/mL)260/280260/230
9/12 SAMmt-YCPlac1111401.801.90
9/12 T7-RBS-psB1C32901.781.65

9/13

Liquid Culture

Yasuda
SampleMedium
9/12 dddD-pSB1C3CP
9/12 dddD-pSB1C3CP
9/12 dddD-pSB1C3CP
9/12 T7-RBS-HisCP
9/12 T7-RBS-HisCP
9/12 T7-RBS-HisCP
controlCP

9/14

Colony PCR

Yasuda
Master Mix
Quick Taq335
VF13.4
VR13.4
MilliQ308.2
TOTAL670

9/14

Gel Extraction

Honda
LaneDNAvolume
11kbp ladder5
23/24 PCR(dddD)cloning E,S15
39/13SAMmT(control)15
49/13SAMmT(EP)15
59/13Pcon-pSB1C3(control)15
69/13Pcon-pSB1C3(XS)

9/14

Electrophoresis for confirmation

Honda, Li and Yamauchi
LaneRestriction Enzyme Digestion Products
19/12 AT-YcPlac 1~8
29/12 AT-YcPlac 1~8
39/12 AT-YcPlac 1~8
49/12 AT-YcPlac 1~8
59/12 AT-YcPlac 1~8
69/12 AT-YcPlac 1~8
79/12 AT-YcPlac 1~8
89/12 AT-YcPlac 1~8
9GeneRuler
109/12 AT-His-YcPlac 1~8
119/12 AT-His-YcPlac 1~8
129/12 AT-His-YcPlac 1~8
139/12 AT-His-YcPlac 1~8
149/12 AT-His-YcPlac 1~8
159/12 AT-His-YcPlac 1~8
169/12 AT-His-YcPlac 1~8
179/12 AT-His-YcPlac 1~8
LaneRestriction Enzyme Digestion Products
19/12 mamB-YcPlac 1~8
29/12 mamB-YcPlac 1~8
39/12 mamB-YcPlac 1~8
49/12 mamB-YcPlac 1~8
59/12 mamB-YcPlac 1~8
69/12 mamB-YcPlac 1~8
79/12 mamB-YcPlac 1~8
89/12 mamB-YcPlac 1~8
9GeneRuler
109/12 DECARB-YcPlac 1~8
119/12 DECARB-YcPlac 1~8
129/12 DECARB-YcPlac 1~8
139/12 DECARB-YcPlac 1~8
149/12 DECARB-YcPlac 1~8
159/12 DECARB-YcPlac 1~8
169/12 DECARB-YcPlac 1~8
179/12 DECARB-YcPlac 1~8
LaneRestriction Enzyme Digestion Products
19/12 DECARB 9~16
29/12 DECARB 9~16
39/12 DECARB 9~16
49/12 DECARB 9~16
59/12 DECARB 9~16
69/12 DECARB 9~16
79/12 DECARB 9~16
89/12 DECARB 9~16
9GeneRuler-YcPlac
109/12 DECARB-YcPlac 17~20
119/12 DECARB-Ycplac 17~20
129/12 DECARB-Ycplac 17~20
139/12 DiDECARB-YcPlac 1~4
149/12 DiDECARB-YcPlac 1~4
159/12 DiDECARB-YcPlac 1~4
169/12 DiDECARB-YcPlac 1~4
179/12 DiDECARB-YcPlac 1~4
LaneRestriction Enzyme Digestion Products
19/12 DiDECARB-YcPlac 5~12
29/12 DiDECARB-YcPlac 5~12
39/12 DiDECARB-YcPlac 5~12
49/12 DiDECARB-YcPlac 5~12
59/12 DiDECARB-YcPlac 5~12
69/12 DiDECARB-YcPlac 5~12
79/12 DiDECARB-YcPlac 5~12
89/12 DiDECARB-YcPlac 5~12
9GeneRuler
109/12 DiDECARB-YcPlac 13~20
119/12 DiDECARB-YcPlac 13~20
129/12 DiDECARB-YcPlac 13~20
139/12 DiDECARB-YcPlac 13~20
149/12 DiDECARB-YcPlac 13~20
159/12 DiDECARB-YcPlac 13~20
169/12 DiDECARB-YcPlac 13~20
179/12 DiDECARB-YcPlac 13~20

9/14

Miniprep

Honda
DNAdensity(µg/ml)260/280260/230
9/14 dddD(µ)-pSB1C36451.821.70
9/14 dddD(µ)-pSB1C36601.691.74
9/14 dddD(µ)-pSB1C36351.871.57
9/14 T7-RBS-His-pSB1C34401.651.23
9/14 T7-RBS-His-pSB1C34501.921.46
9/14 T7-RBS-His-pSB1C31901.ss1.26

9/14

Restriction Enzyme Digestion

Honda
SampleVolumeSpe1Pst1Xba1)BuffurBSAMilliQTOTAL
9/14 dddD(µ)-pSB1C360226(M)63860
9/14 dddD(µ)-pSB1C360226(M)63860
9/14 dddD(µ)-pSB1C360226(M)63860
9/14 T7-RBS-His-pSB1C351103(H)01830
9/14 T7-RBS-His-pSB1C351103(H)01830
9/14 T7-RBS-His-pSB1C3101103(H)01530

9/14

Ligation

Yasuda
NEB T4DNALigase10XLig Buffer9/12 DECARB XS column9/14 Pcon-pSB1C3 XS DephosphorylatedMilliQTOTAL
122.930.8313.2420
NEB T4DNALigase10XLig Buffer9/12 DiDECARB XS column9/14 Pcon-pSB1C3 XS DephosphorylatedMilliQTOTAL
123.660.8312.520

Retrieved from "http://2014.igem.org/Team:Kyoto/Notebook/DMS_Synthesis"