Template:Kyoto/Notebook/Sep 6

From 2013.igem.org

(Difference between revisions)
(→Colony PCR)
(→Liquid Culture)
 
(44 intermediate revisions not shown)
Line 1: Line 1:
==Sep 6==
==Sep 6==
 +
===Electrophoresis===
 +
<div class="experiment">
 +
<span class="author">Hirano</span>
 +
{| class="wikitable"
 +
!Lane||Sample
 +
|-
 +
|1||100bp ladder
 +
|-
 +
|2||9/4 RBS-lysis3-DT -3
 +
|-
 +
|3||9/4 RBS-lysis3-DT -4
 +
|-
 +
|4||9/4 RBS-lysis3-DT -5
 +
|-
 +
|5||9/4 RBS-lysis1-DT -3
 +
|-
 +
|6||9/4 Ptrc-KaiC -1
 +
|-
 +
|7||9/4 pSB4K5 -1
 +
|-
 +
|8||100bp ladder
 +
|}
 +
[[File:Igku Sep6 ColonyPCRN1.jpg]]<br>
 +
</div>
 +
 +
===Colony PCR===
 +
<div class="experiment">
 +
<span class="author">No name</span>
 +
{| class="wikitable"
 +
!|Sample||base pair
 +
|-
 +
|Pconst-spinach-DT-(1~4)||563
 +
|-
 +
|Plac-spinach-DT-(1~4)||659
 +
|-
 +
|Pcon-pT181 antisense-(1~4)||468
 +
|-
 +
|Ptet-pT181 antisense-(1~4)||604
 +
|-
 +
|Pconst-pT181 attenuator-(1~4)||568
 +
|-
 +
|Pconst+RBS-tetR-DT-(1~4)||1121
 +
|-
 +
|Ptet+RBS-lacZ&alpha;-DT-(1~4)||1162
 +
|}
 +
{| class="wikitable"
 +
!PreDenature||Denature||Annealing||Extension||cycle
 +
|-
 +
|94&deg;C||94&deg;C||55&deg;C||68&deg;C||--
 +
|-
 +
|5min||30s||30s||40s||30cycles
 +
|}
 +
 +
</div>
 +
===Liquid Culture===
===Liquid Culture===
<div class="experiment">
<div class="experiment">
Line 6: Line 61:
!Sample||medium
!Sample||medium
|-
|-
-
|9/4 RBS-Lysis3＋DT3||Plusgrow medium(+Cp)
+
|9/4 RBS-Lysis3+DT3||Plusgrow medium(+CP)
|-
|-
-
|9/4 RBS-Lysis1＋DT3||Plusgrow medium(+Cp)
+
|9/4 RBS-Lysis1+DT3||Plusgrow medium(+CP)
|-
|-
-
|9/4 PSB4K5||Plusgrow medium(+K)　
+
|9/4 pSB4K5||Plusgrow medium(+Kan)
|}
|}
</div>
</div>
-
===Colony PCR===
+
===Electrophoresis===
<div class="experiment">
<div class="experiment">
<span class="author">No name</span>
<span class="author">No name</span>
{| class="wikitable"
{| class="wikitable"
-
!|Sample||base pair
+
!Lane||Sample
|-
|-
-
|Pconst-spinach-DT-(1)||-
+
|1||100bp ladder
|-
|-
-
|Pconst-spinach-DT-(2)||-
+
|2||Pconst-spinach-DT -(1)
|-
|-
-
|Pconst-spinach-DT-(3)||-
+
|3||Pconst-spinach-DT -(2)
|-
|-
-
|Pconst-spinach-DT-(4)||-
+
|4||Pconst-spinach-DT -(3)
|-
|-
-
|Plac-spinach-DT-(1)||-
+
|5||Pconst-spinach-DT -(4)
|-
|-
-
|Plac-spinach-DT-(2)||-
+
|6||Plac-spinach-DT -(1)
|-
|-
-
|Plac-spinach-DT-(3)||-
+
|7||Plac-spinach-DT -(2)
|-
|-
-
|Plac-spinach-DT-(4)||-
+
|8||Plac-spinach-DT -(3)
|-
|-
-
|Pcon-PT181 antisense-(1)||-
+
|9||Plac-spinach-DT -(4)
|-
|-
-
|Pcon-PT181 antisense-(2)||-
+
|10||Pcon-pT181 antisense -(1)
|-
|-
-
|Pcon-PT181 antisense-(3)||-
+
|11||Pcon-pT181 antisense -(2)
|-
|-
-
|Pcon-PT181 antisense-(4)||-
+
|12||Pcon-pT181 antisense -(3)
 +
|-
 +
|13||Pcon-pT181 antisense -(4)
|}
|}
{| class="wikitable"
{| class="wikitable"
-
!|Sample||base pair
+
!Lane||Sample
|-
|-
-
|Ptet-PT181 antisense-(1)||-
+
|1||100bp ladder
|-
|-
-
|Ptet-PT181 antisense-(2)||-
+
|2||Ptet-pT181 antisense -(1)
|-
|-
-
|Ptet-PT181 antisense-(3)||-
+
|3||Ptet-pT181 antisense -(2)
|-
|-
-
|Ptet-PT181 antisense-(4)||-
+
|4||Ptet-pT181 antisense -(3)
|-
|-
-
|Plac-PT181 attenuater-(1)||-
+
|5||Ptet-pT181 antisense -(4)
|-
|-
-
|Plac-PT181 attenuater-(2)||-
+
|6||Plac-pT181 attenuator -(1)
|-
|-
-
|Plac-PT181 attenuater-(3)||-
+
|7||Plac-pT181 attenuator -(2)
|-
|-
-
|Plac-PT181 attenuater-(4)||-
+
|8||Plac-pT181 attenuator -(3)
 +
|-
 +
|9||Plac-pT181 attenuator -(4)
|}
|}
 +
[[File:igku_Sep6_COlonyPCRN1_1a.jpg]]<br>
 +
[[File:igku_Sep6_colonyPCR_N2_1b.jpg]]<br>
 +
</div>
===Miniprep===
===Miniprep===
Line 71: Line 133:
!DNA||concentration [&micro;g/mL]||260/280||260/230
!DNA||concentration [&micro;g/mL]||260/280||260/230
|-
|-
-
|PT181 antisense-(1)||238.7||1.68||1.44
+
|pT181 antisense-(1)||238.7||1.68||1.44
|-
|-
-
|PT181 antisense-(2)||248.8||1.83||1.77
+
|pT181 antisense-(2)||248.8||1.83||1.77
|-
|-
-
|PT181 antisense-(3)||223.2||1.78||1.61
+
|pT181 antisense-(3)||223.2||1.78||1.61
|-
|-
-
|PT181 antisense-(4)||220.8||1.89||1.92
+
|pT181 antisense-(4)||220.8||1.89||1.92
|-
|-
|tetR aptamer-(1)||252.5||1.89||1.90
|tetR aptamer-(1)||252.5||1.89||1.90
Line 83: Line 145:
|tetR aptamer-(2)||211.2||1.90||1.99
|tetR aptamer-(2)||211.2||1.90||1.99
|-
|-
-
|DT181 attenuater||102.2||1.98||2.17
+
|DT181 attenuator||102.2||1.98||2.17
|-
|-
|121R aptamer-DT||251.7||1.92||2.01
|121R aptamer-DT||251.7||1.92||2.01
|-
|-
-
|PSB4K5||212.9||1.80||1.90
+
|pSB4K5||212.9||1.80||1.90
|-
|-
-
|RBS-lacZα-DT||185.7||1.78||1.79
+
|RBS-lacZ&alpha;-DT||185.7||1.78||1.79
|-
|-
|RBS-lysis2-DT-(1)||84.8||1.85||2.24
|RBS-lysis2-DT-(1)||84.8||1.85||2.24
Line 99: Line 161:
<span class="author">Tatsui</span>
<span class="author">Tatsui</span>
{| class="wikitable"
{| class="wikitable"
-
! ||9/6 RBS-lysis2-DT (78.3 µg/mL)||EcoRI||SpeI||XbaI||PstI||BSA||Buffer||MilliQ||total
+
! ||9/6 RBS-lysis2-DT (78.3 &micro;g/mL)||XbaI||PstI||BSA||Buffer||MilliQ||total
|-
|-
-
|2 cuts||22&micro;L||0&micro;L||0&micro;L||1&micro;L||1&micro;L||3&micro;L||3&micro;L||0&micro;L||30&micro;L
+
|2 cuts||22 &micro;L||1 &micro;L||1 &micro;L||3 &micro;L||3 &micro;L||0 &micro;L||30 &micro;L
|-
|-
-
|NC||1.3&micro;L||0&micro;L||0&micro;L||0&micro;L||0&micro;L||1&micro;L||1&micro;L||6.7&micro;L||10&micro;L
+
|NC||1.3 &micro;L||0 &micro;L||0 &micro;L||1 &micro;L||1 &micro;L||6.7 &micro;L||10 &micro;L
|}
|}
-
*at 37&deg;C, for 1h
 
{| class="wikitable"
{| class="wikitable"
-
! ||aptamer-DT (251.7 µg/mL)||EcoRI||SpeI||XbaI||PstI||BSA||Buffer||MilliQ||total
+
! ||aptamer-DT (251.7 &micro;g/mL)||XbaI||PstI||BSA||Buffer||MilliQ||total
|-
|-
-
|2 cuts||0.4&micro;L||0&micro;L||0&micro;L||1&micro;L||1&micro;L||3&micro;L||3&micro;L||14.1&micro;L||30&micro;L
+
|2 cuts||0.4 &micro;L||1 &micro;L||1 &micro;L||3 &micro;L||3 &micro;L||14.1 &micro;L||30 &micro;L
|-
|-
-
|NC||0.4&micro;L||0&micro;L||0&micro;L||0&micro;L||0&micro;L||1&micro;L||1&micro;L||7.6&micro;L||10&micro;L
+
|NC||0.4 &micro;L||0 &micro;L||0 &micro;L||1 &micro;L||1 &micro;L||7.6 &micro;L||10 &micro;L
|}
|}
-
*at 37&deg;C, for 1h
 
{| class="wikitable"
{| class="wikitable"
-
! ||Pλ-luxI (86.4 µg/mL)||EcoRI||SpeI||XbaI||PstI||BSA||Buffer||MilliQ||total
+
! ||P&lambda;-luxI (86.4 &micro;g/mL)||EcoRI||SpeI||BSA||Buffer||MilliQ||total
|-
|-
-
|2 cuts||23.1&micro;L||1&micro;L||1&micro;L||0&micro;L||0&micro;L||3&micro;L||3&micro;L||1.9&micro;L||30&micro;L
+
|2 cuts||23.1 &micro;L||1 &micro;L||1 &micro;L||3 &micro;L||3 &micro;L||1.9 &micro;L||30 &micro;L
|-
|-
-
|NC||1.2&micro;L||0&micro;L||0&micro;L||0&micro;L||0&micro;L||1&micro;L||1&micro;L||6.8&micro;L||10&micro;L
+
|NC||1.2 &micro;L||0 &micro;L||0 &micro;L||1 &micro;L||1 &micro;L||6.8 &micro;L||10 &micro;L
|}
|}
-
*at 37&deg;C, for 1h
+
*at 37 &deg;C, for 1h
-
===Colony PCR===
+
===Electrophoresis===
<div class="experiment">
<div class="experiment">
<span class="author">Hirano</span>
<span class="author">Hirano</span>
{| class="wikitable"
{| class="wikitable"
-
!|Sample||base pair
+
!Lane||Sample
 +
|-
 +
|1||100bp ladder
 +
|-
 +
|2||Pconst+RBS-tetR-DT -(1)
 +
|-
 +
|3||Pconst+RBS-tetR-DT -(2)
|-
|-
-
|Pconst＋RBS-tetR-DT-(1)||1121
+
|4||Pconst+RBS-tetR-DT -(3)
|-
|-
-
|Pconst＋RBS-tetR-DT-(2)||1121
+
|5||Pconst+RBS-tetR-DT -(4)
|-
|-
-
|Pconst＋RBS-tetR-DT-(3)||1121
+
|6||Ptet+RBS-lacZ&alpha;-DT -(1)
|-
|-
-
|Pconst＋RBS-tetR-DT-(4)||1121
+
|7||Ptet+RBS-lacZ&alpha;-DT -(2)
|-
|-
-
|Ptet＋RBS-lacZα-DT-(1)||1162
+
|8||100bp ladder
|-
|-
-
|Ptet＋RBS-lacZα-DT-(2)||1162
+
|9||Ptet+RBS-lacZ&alpha;-DT -(3)
|-
|-
-
|Ptet＋RBS-lacZα-DT-(3)||1162
+
|10||Ptet+RBS-lacZ&alpha;-DT -(4)
|-
|-
-
|Ptet＋RBS-lacZα-DT-(4)||1162
+
|11||100bp ladder
|}
|}
 +
[[File:igku_Sep6_electrophoresis_N3.jpg]]<br>
</div>
</div>
Line 157: Line 224:
|1||100bp ladder||--
|1||100bp ladder||--
|-
|-
-
|2||rowspan="2"|8/20 RBS-lysis1-(1)||rowspan="2"|EcoRI & SpeI
+
|2||rowspan="2"|RBS-lysis2-DT||rowspan="2"|XbaI & PstI
|-
|-
|3
|3
Line 163: Line 230:
|4||--||--
|4||--||--
|-
|-
-
|5||rowspan="2"|8/28 RBS-GFP-DT-(2)||rowspan="2"|XbaI & PstI
+
|5||rowspan="2"|aptamer 12_1R-DT||rowspan="2"|XbaI & PstI
|-
|-
|6
|6
|}
|}
-
[[File:igku_xxbeforexx.xxx]]<br>
 
-
[[File:igku_xxafterxx.xxx]]<br>
 
{| class="wikitable"
{| class="wikitable"
!Lane||DNA||Enzyme
!Lane||DNA||Enzyme
Line 174: Line 239:
|1||100bp ladder||--
|1||100bp ladder||--
|-
|-
-
|2||rowspan="2"|8/28 Plux||rowspan="2"|SpeI & PstI
+
|2||rowspan="2"|P&lambda;-luxI||rowspan="2"|EcoRI & SpeI
|-
|-
|3
|3
-
|-
 
-
|4||--||--
 
-
|-
 
-
|5||rowspan="2"|8/20 RBS-tetR-DT-(1)||rowspan="2"|XbaI & PstI
 
-
|-
 
-
|6
 
|}
|}
-
[[File:igku_xxbeforexx.xxx]]<br>
+
[[File:igku_Sep6_gel_extraction_N4_2.jpg]]<br>
-
[[File:igku_xxafterxx.xxx]]<br>
+
[[File:igku_Sep6_gel_extraction_N4_3.jpg]]<br>
{| class="wikitable"
{| class="wikitable"
!Name||concentration[&micro;g/mL]||260/280||260/230
!Name||concentration[&micro;g/mL]||260/280||260/230
|-
|-
-
|Pcon-RBS-GFP-DT (EcoRI&SpeI)||7.1||1.88||0.07
+
|RBS-lysis2-DT (XbaI & PstI)||7.1||1.88||0.07
|-
|-
-
|Pcon-RBS-luxR-DT (EcoRI&XbaI)||15.3||1.93||0.70
+
|aptamer 12_1R-DT (XbaI & PstI)||15.3||1.93||0.70
|-
|-
-
|Plux (SpeI&PstI)||4.5||1.79||0.31
+
|P&lambda;-luxI (EcoRI & SpeI)||4.5||1.79||0.31
-
|-
+
-
|RBS-GFP-DT (XbaI&PstI)||6.0||2.16||0.03
+
-
|-
+
-
|RBS-tetR-DT (XbaI&PstI)||7.6||1.91||0.38
+
|}
|}
 +
[[File:igku_Sep6_gel_extraction_N5_1.jpg]]<br>
 +
[[File:igku_Sep6_gel_extraction_N5_2.jpg]]<br>
</div>
</div>
-
 
===Liquid Culture===
===Liquid Culture===
Line 208: Line 264:
!Sample||medium
!Sample||medium
|-
|-
-
|8/21 RBS-Lysis2-9||Plusgrow medium(+Amp)
+
|9/5 Pconst-spinach-DT-(1)||Plusgrow medium(+Amp&CP)
|-
|-
-
|8/21 RBS-Lysis2-12||Plusgrow medium(+Amp)
+
|9/5 Ptet-pT181 antisense-(1)||Plusgrow medium(+CP)
|-
|-
-
|8/21 RBS-Lysis2-14||Plusgrow medium(+Amp)　
+
|9/5 Pconst+RBS-tetR-DT-(1)||Plusgrow medium(+Amp&CP)
-
|}
+
-
</div>
+
-
 
+
-
===Colony PCR===
+
-
<div class="experiment">
+
-
<span class="author">No name</span>
+
-
{| class="wikitable"
+
-
!|Sample||base pair
+
|-
|-
-
|8/27 Pcon-pT181 antisense-(1)||542
+
|9/5 Ptet+RBS-lacZ&alpha;-DT-(1)||Plusgrow medium(+CP)
|-
|-
-
|8/26 pT181 attenuator-DT-(1)||738
+
|9/5 Pcon-pT181 attenuator-(1)||Plusgrow medium(+CP)
-
|}
+
-
{| class="wikitable"
+
-
!PreDenature||Denature||Annealing||Extension||cycle
+
|-
|-
-
|94&deg;C||94&deg;C||55&deg;C||68&deg;C||--
+
|9/4 pT181 antisense-(1)||Plusgrow medium(+CP)
|-
|-
-
|5min||30s||30s||30s||30cycles
+
|9/4 aptamer 12_1R-(1)||Plusgrow medium(+CP)
|}
|}
-
[[File:igku_Aug19electrophoresis1.png]]
 
</div>
</div>
Line 242: Line 286:
!|Sample||base pair
!|Sample||base pair
|-
|-
-
|8/27 Pcon-pT181 antisense-(1)||542
+
|9/5 Plac-spinach-DT-(5~12)||563
 +
|-
 +
|9/5 Pcon-antisense-(5~12)||468
 +
|-
 +
|9/5 Plac-attenuator-(5~12)||664
|-
|-
-
|8/26 pT181 attenuator-DT-(1)||738
+
|9/5 pT181 antisense+Plac-(1)||-
|}
|}
{| class="wikitable"
{| class="wikitable"
Line 251: Line 299:
|94&deg;C||94&deg;C||55&deg;C||68&deg;C||--
|94&deg;C||94&deg;C||55&deg;C||68&deg;C||--
|-
|-
-
|5min||30s||30s||30s||30cycles
+
|5min||30s||30s||42s||30cycles
|}
|}
-
[[File:igku_Aug19electrophoresis1.png]]
 
</div>
</div>

Latest revision as of 03:56, 28 September 2013

Contents

Sep 6

Electrophoresis

Hirano

LaneSample
1100bp ladder
29/4 RBS-lysis3-DT -3
39/4 RBS-lysis3-DT -4
49/4 RBS-lysis3-DT -5
59/4 RBS-lysis1-DT -3
69/4 Ptrc-KaiC -1
79/4 pSB4K5 -1
8100bp ladder

Igku Sep6 ColonyPCRN1.jpg

Colony PCR

No name

Samplebase pair
Pconst-spinach-DT-(1~4)563
Plac-spinach-DT-(1~4)659
Pcon-pT181 antisense-(1~4)468
Ptet-pT181 antisense-(1~4)604
Pconst-pT181 attenuator-(1~4)568
Pconst+RBS-tetR-DT-(1~4)1121
Ptet+RBS-lacZα-DT-(1~4)1162
PreDenatureDenatureAnnealingExtensioncycle
94°C94°C55°C68°C--
5min30s30s40s30cycles

Liquid Culture

No name

Samplemedium
9/4 RBS-Lysis3+DT3Plusgrow medium(+CP)
9/4 RBS-Lysis1+DT3Plusgrow medium(+CP)
9/4 pSB4K5Plusgrow medium(+Kan)

Electrophoresis

No name

LaneSample
1100bp ladder
2Pconst-spinach-DT -(1)
3Pconst-spinach-DT -(2)
4Pconst-spinach-DT -(3)
5Pconst-spinach-DT -(4)
6Plac-spinach-DT -(1)
7Plac-spinach-DT -(2)
8Plac-spinach-DT -(3)
9Plac-spinach-DT -(4)
10Pcon-pT181 antisense -(1)
11Pcon-pT181 antisense -(2)
12Pcon-pT181 antisense -(3)
13Pcon-pT181 antisense -(4)
LaneSample
1100bp ladder
2Ptet-pT181 antisense -(1)
3Ptet-pT181 antisense -(2)
4Ptet-pT181 antisense -(3)
5Ptet-pT181 antisense -(4)
6Plac-pT181 attenuator -(1)
7Plac-pT181 attenuator -(2)
8Plac-pT181 attenuator -(3)
9Plac-pT181 attenuator -(4)

Igku Sep6 COlonyPCRN1 1a.jpg
Igku Sep6 colonyPCR N2 1b.jpg

Miniprep

Honda and Kojima

DNAconcentration [µg/mL]260/280260/230
pT181 antisense-(1)238.71.681.44
pT181 antisense-(2)248.81.831.77
pT181 antisense-(3)223.21.781.61
pT181 antisense-(4)220.81.891.92
tetR aptamer-(1)252.51.891.90
tetR aptamer-(2)211.21.901.99
DT181 attenuator102.21.982.17
121R aptamer-DT251.71.922.01
pSB4K5212.91.801.90
RBS-lacZα-DT185.71.781.79
RBS-lysis2-DT-(1)84.81.852.24

Restriction Enzyme Digestion

Tatsui

9/6 RBS-lysis2-DT (78.3 µg/mL)XbaIPstIBSABufferMilliQtotal
2 cuts22 µL1 µL1 µL3 µL3 µL0 µL30 µL
NC1.3 µL0 µL0 µL1 µL1 µL6.7 µL10 µL
aptamer-DT (251.7 µg/mL)XbaIPstIBSABufferMilliQtotal
2 cuts0.4 µL1 µL1 µL3 µL3 µL14.1 µL30 µL
NC0.4 µL0 µL0 µL1 µL1 µL7.6 µL10 µL
Pλ-luxI (86.4 µg/mL)EcoRISpeIBSABufferMilliQtotal
2 cuts23.1 µL1 µL1 µL3 µL3 µL1.9 µL30 µL
NC1.2 µL0 µL0 µL1 µL1 µL6.8 µL10 µL
  • at 37 °C, for 1h

Electrophoresis

Hirano

LaneSample
1100bp ladder
2Pconst+RBS-tetR-DT -(1)
3Pconst+RBS-tetR-DT -(2)
4Pconst+RBS-tetR-DT -(3)
5Pconst+RBS-tetR-DT -(4)
6Ptet+RBS-lacZα-DT -(1)
7Ptet+RBS-lacZα-DT -(2)
8100bp ladder
9Ptet+RBS-lacZα-DT -(3)
10Ptet+RBS-lacZα-DT -(4)
11100bp ladder

Igku Sep6 electrophoresis N3.jpg

Gel Extraction

No name

LaneDNAEnzyme
1100bp ladder--
2RBS-lysis2-DTXbaI & PstI
3
4----
5aptamer 12_1R-DTXbaI & PstI
6
LaneDNAEnzyme
1100bp ladder--
2Pλ-luxIEcoRI & SpeI
3

Igku Sep6 gel extraction N4 2.jpg
Igku Sep6 gel extraction N4 3.jpg

Nameconcentration[µg/mL]260/280260/230
RBS-lysis2-DT (XbaI & PstI)7.11.880.07
aptamer 12_1R-DT (XbaI & PstI)15.31.930.70
Pλ-luxI (EcoRI & SpeI)4.51.790.31

Igku Sep6 gel extraction N5 1.jpg
Igku Sep6 gel extraction N5 2.jpg

Liquid Culture

No name

Samplemedium
9/5 Pconst-spinach-DT-(1)Plusgrow medium(+Amp&CP)
9/5 Ptet-pT181 antisense-(1)Plusgrow medium(+CP)
9/5 Pconst+RBS-tetR-DT-(1)Plusgrow medium(+Amp&CP)
9/5 Ptet+RBS-lacZα-DT-(1)Plusgrow medium(+CP)
9/5 Pcon-pT181 attenuator-(1)Plusgrow medium(+CP)
9/4 pT181 antisense-(1)Plusgrow medium(+CP)
9/4 aptamer 12_1R-(1)Plusgrow medium(+CP)

Colony PCR

No name

Samplebase pair
9/5 Plac-spinach-DT-(5~12)563
9/5 Pcon-antisense-(5~12)468
9/5 Plac-attenuator-(5~12)664
9/5 pT181 antisense+Plac-(1)-
PreDenatureDenatureAnnealingExtensioncycle
94°C94°C55°C68°C--
5min30s30s42s30cycles
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