Team:CU-Boulder/Test
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+ | <div id="rightscroll"> | ||
+ | <html><div id="detect_Saturday_13th_July.html"></div></html> | ||
+ | <html> | ||
+ | <div class ="tbnote" style="background: url('http://thesmashable.com/wp-content/uploads/2012/06/james-bond-skyfall-007-wallpapers-desktop-backgrounds-james-bond-hd-wallpapers-007-2012-11.jpg'); background-size: 50%; background-opacity: 0.4;> | ||
+ | <h2>Detect</h2> | ||
+ | <a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Detect" target="_blank" class="tbnotelogo PSlogo"> ASDF </a> | ||
+ | <div style="clear: both;"></div> | ||
+ | <h3>Saturday 13<sup>th</sup> July</h3> | ||
+ | <p><b><em> | ||
+ | <!-- === Modify from here === --> | ||
+ | PCR circular and linear, Conjugation of XL-10 (with sSP011), Patches, Digestion M13 backbone, RFP insert, Gel of PCR | ||
+ | <!-- === To here === --> | ||
+ | </br></em></b></p> | ||
+ | <!-- === Modify from here === --> | ||
+ | <b>PCR circular and linear, 40.5°C, 60°</b> | ||
+ | of pSB1A3, pSB1C3<br> | ||
+ | <br> | ||
+ | <TABLE BORDER> | ||
+ | <TR><TD><b>Reagent</b></TD><TD><b>Volume</b></TD></TR> | ||
+ | <TR><TD></TD><TD><b>1x</b></TD></TR> | ||
+ | <TR><TD>Nuclease-free water</TD><TD>37.25 ul</TD></TR> | ||
+ | <TR><TD>5x Phusion HF Buffer</TD><TD>10 ul</TD></TR> | ||
+ | <TR><TD>10 mM dNTPs</TD><TD>1 ul</TD></TR> | ||
+ | <TR><TD>Forward Primer (10 uM)</TD><TD>0.5 ul</TD></TR> | ||
+ | <TR><TD>Reverse Primer (10 uM)</TD><TD>0.5 ul</TD></TR> | ||
+ | <TR><TD>Template Plasmid</TD><TD>0.25 ul</TD></TR> | ||
+ | <TR><TD>Phusion DNA Polymerase</TD><TD>0.5 ul</TD></TR> | ||
+ | <TR><TD><b>Total Volume</b></TD><TD>50 ul</TD></TR> | ||
+ | </TABLE><br> | ||
+ | <br> | ||
+ | <TABLE BORDER> | ||
+ | <TR><TD><b>Thermocycler Protocol: NEB Phusion</b></TD><TD> </TD><TD> </TD><TD> </TD><TD> </TD></TR> | ||
+ | <TR><TD> </TD><TD>Temp</TD><TD>Time</TD><TD> </TD><TD> </TD></TR> | ||
+ | <TR><TD>Start</TD><TD>98°C</TD><TD>30 sec</TD><TD>Melt</TD><TD> </TD></TR> | ||
+ | <TR><TD> </TD><TD> </TD><TD> </TD><TD> </TD><TD> </TD></TR> | ||
+ | <TR><TD>Cycle 1</TD><TD>98°C</TD><TD>5 sec</TD><TD>Melt</TD><TD> 35 cycles </TD></TR> | ||
+ | <TR><TD>Cycle 2</TD><TD>40.5°C / 60°C </TD><TD>25 sec</TD><TD>Anneal</TD><TD></TD></TR> | ||
+ | <TR><TD>Cycle 3</TD><TD>72°C</TD><TD>5 min</TD><TD>Extend</TD><TD> </TD></TR> | ||
+ | <TR><TD> </TD><TD> </TD><TD> </TD><TD> </TD><TD> </TD></TR> | ||
+ | <TR><TD>Finish</TD><TD>72°C</TD><TD>5 min</TD><TD>Extend</TD><TD> </TD></TR> | ||
+ | <TR><TD>Store</TD><TD>10°C</TD><TD>Forever</TD><TD>Store</TD><TD> </TD></TR> | ||
+ | </TABLE><br> | ||
+ | <br> | ||
+ | |||
+ | <b>Conjugation of XL-10 (with sSP011)</b><br> | ||
+ | 1) From O/N cultures Dilute strains 1/100 in LB<br> | ||
+ | 2) Wait for OD to reach O,2<br> | ||
+ | 3) Prepare tube (in BD tubes) :<br> | ||
+ | - Tube = 0,5mL LB with Strain (sSP011) + 0,5mL LB with Strain (XL-10 Kan)<br> | ||
+ | 4) Incubate 2 hours at 37°C (actually not in the shaker, but we accidently kept them in the shaker...)<br> | ||
+ | 5) Plate 20ul for mixed tube on LB antiobiotics (Tet, Kan)<br> | ||
+ | 6) Incubate overnight at 37°C<br> | ||
+ | <br> | ||
+ | <br> | ||
+ | <b>Patches</b><br> | ||
+ | check and make new ones<br> | ||
+ | <br> | ||
+ | <br> | ||
+ | <b>Digestion M13 backbone, RFP insert</b><br> | ||
+ | for Backbone (M13mp18 plasmid): 3ug<br> | ||
+ | 7,58 ul plasmid (c=395ng/ul)<br> | ||
+ | 3 ul EcoRI<br> | ||
+ | 3 ul PstI<br> | ||
+ | 3ul 10x Fast Digest<br> | ||
+ | 13,42 ul H20<br> | ||
+ | incubate for 12 min on 37°<br> | ||
+ | heat inactivation: 80° 5 min<br> | ||
+ | for insert (BBa_J04450): 5ug<br> | ||
+ | 31,4 ul plasmid<br> | ||
+ | 5 ul EcoRi<br> | ||
+ | 5 ul PstI<br> | ||
+ | 3,6 ul H20<br> | ||
+ | incubate for 20 min at 37°<br> | ||
+ | heat inactivation: 80° 5min<br> | ||
+ | <br> | ||
+ | <b>Gel of PCR </b> (Amp 40.5 circ, lin, Chl 60° lin, circ)<br> | ||
+ | 100V, 20 min 1% gel<br> | ||
+ | |||
+ | <!-- === To here === --> | ||
+ | </div> | ||
+ | </html> | ||
+ | </div> | ||
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Latest revision as of 20:20, 1 July 2014
-
DAY NOTES
- DETECT
- TARGET
- INFILTRATE
- SABOTAGE
ASDF
Conjugation of XL-10 (with sSP011)
1) From O/N cultures Dilute strains 1/100 in LB
2) Wait for OD to reach O,2
3) Prepare tube (in BD tubes) :
- Tube = 0,5mL LB with Strain (sSP011) + 0,5mL LB with Strain (XL-10 Kan)
4) Incubate 2 hours at 37°C (actually not in the shaker, but we accidently kept them in the shaker...)
5) Plate 20ul for mixed tube on LB antiobiotics (Tet, Kan)
6) Incubate overnight at 37°C
Patches
check and make new ones
Digestion M13 backbone, RFP insert
for Backbone (M13mp18 plasmid): 3ug
7,58 ul plasmid (c=395ng/ul)
3 ul EcoRI
3 ul PstI
3ul 10x Fast Digest
13,42 ul H20
incubate for 12 min on 37°
heat inactivation: 80° 5 min
for insert (BBa_J04450): 5ug
31,4 ul plasmid
5 ul EcoRi
5 ul PstI
3,6 ul H20
incubate for 20 min at 37°
heat inactivation: 80° 5min
Gel of PCR (Amp 40.5 circ, lin, Chl 60° lin, circ)
100V, 20 min 1% gel
</div>
Saturday 13th July
PCR circular and linear, Conjugation of XL-10 (with sSP011), Patches, Digestion M13 backbone, RFP insert, Gel of PCR
PCR circular and linear, 40.5°C, 60° of pSB1A3, pSB1C3Reagent | Volume |
1x | |
Nuclease-free water | 37.25 ul |
5x Phusion HF Buffer | 10 ul |
10 mM dNTPs | 1 ul |
Forward Primer (10 uM) | 0.5 ul |
Reverse Primer (10 uM) | 0.5 ul |
Template Plasmid | 0.25 ul |
Phusion DNA Polymerase | 0.5 ul |
Total Volume | 50 ul |
Thermocycler Protocol: NEB Phusion | ||||
Temp | Time | |||
Start | 98°C | 30 sec | Melt | |
Cycle 1 | 98°C | 5 sec | Melt | 35 cycles |
Cycle 2 | 40.5°C / 60°C | 25 sec | Anneal | |
Cycle 3 | 72°C | 5 min | Extend | |
Finish | 72°C | 5 min | Extend | |
Store | 10°C | Forever | Store |
Conjugation of XL-10 (with sSP011)
1) From O/N cultures Dilute strains 1/100 in LB
2) Wait for OD to reach O,2
3) Prepare tube (in BD tubes) :
- Tube = 0,5mL LB with Strain (sSP011) + 0,5mL LB with Strain (XL-10 Kan)
4) Incubate 2 hours at 37°C (actually not in the shaker, but we accidently kept them in the shaker...)
5) Plate 20ul for mixed tube on LB antiobiotics (Tet, Kan)
6) Incubate overnight at 37°C
Patches
check and make new ones
Digestion M13 backbone, RFP insert
for Backbone (M13mp18 plasmid): 3ug
7,58 ul plasmid (c=395ng/ul)
3 ul EcoRI
3 ul PstI
3ul 10x Fast Digest
13,42 ul H20
incubate for 12 min on 37°
heat inactivation: 80° 5 min
for insert (BBa_J04450): 5ug
31,4 ul plasmid
5 ul EcoRi
5 ul PstI
3,6 ul H20
incubate for 20 min at 37°
heat inactivation: 80° 5min
Gel of PCR (Amp 40.5 circ, lin, Chl 60° lin, circ)
100V, 20 min 1% gel
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