Team:Paris Bettencourt/Notebook/Phage Sensor/Wednesday 7th August.html

From 2013.igem.org

(Difference between revisions)
(Created page with "<html> <div class ="tbnote"> <h2>Phage Sensor</h2> <a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Phage_Sensor" target="_blank" class="tbnotelogo PSlogo"> ASDF </a>...")
 
(2 intermediate revisions not shown)
Line 11: Line 11:
</br></em></b></p>
</br></em></b></p>
<!-- === Modify from here === -->
<!-- === Modify from here === -->
-
<b>Glycerol Stock of sSP008-sSP0011</b><br>
+
<br><b>Glycerol Stock of sSP008-sSP0011</b><br>
Take 1.5ml of ON culture + 500ul Glycerol (60%)<br>
Take 1.5ml of ON culture + 500ul Glycerol (60%)<br>
Freeze at -80°C<br>
Freeze at -80°C<br>
Line 26: Line 26:
<b>Liquid cultures</b> of pir+ E.coli and pKD13 from Jake<br>
<b>Liquid cultures</b> of pir+ E.coli and pKD13 from Jake<br>
<br>
<br>
-
<b>PCR of SPCR4, SPCR7, SPCR8, SPCR9, SPCR10 from circuar backbone, directly taken from Biobrick plate</b>
+
<b>PCR of SPCR4, SPCR7, SPCR8, SPCR9, SPCR10 from circuar backbone, directly taken from Biobrick plate</b><br><br>
-
<br>
+
Protocol<br>
Protocol<br>
<br>
<br>
-
 
<TABLE BORDER>
<TABLE BORDER>
<TR><TD><b>Reagent</b></TD><TD><b>Volume</b></TD></TR>
<TR><TD><b>Reagent</b></TD><TD><b>Volume</b></TD></TR>
<TR><TD> </TD><TD><b>1x</b></TD></TR>
<TR><TD> </TD><TD><b>1x</b></TD></TR>
<TR><TD>Nuclease-free water</TD><TD>37,25µL</TD></TR>
<TR><TD>Nuclease-free water</TD><TD>37,25µL</TD></TR>
-
<TR><5x Phusion HF Buffer</TD><TD>10µL</TD></TR>
+
<TR><TD>5x Phusion HF Buffer</TD><TD>10µL</TD></TR>
<TR><TD>10 mM dNTPs</TD><TD>1µL</TD></TR>
<TR><TD>10 mM dNTPs</TD><TD>1µL</TD></TR>
<TR><TD>Forward Primer (10 µM)</TD><TD>0.5µL</TD></TR>
<TR><TD>Forward Primer (10 µM)</TD><TD>0.5µL</TD></TR>
Line 43: Line 41:
<TR><TD><b>Total Volume</b></TD><TD>50µL</TD></TR>
<TR><TD><b>Total Volume</b></TD><TD>50µL</TD></TR>
</TABLE>
</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>30 sec per kb</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>Transformation</b> Biobricks BBa_K649304 (with pSB1C3 backbone) and BBa_J04450 (with pSB1A3 backbone) into NEB Turbo (heat shock trafo)<br>
 +
<br>
 +
1) Thaw competent cells on ice.<br>
 +
2) Place 20 ul of cells in a pre-chilled Eppendorf tube.<br>
 +
3) Add 2.5 ul of plasmid (from Biobrick stock)<br>
 +
4) Mix gently by flicking the tube.<br>
 +
5) Chill on ice for 10 minutes.<br>
 +
4) Heat shock at 42 °C for 30 seconds <br>
 +
5) Return to ice for 2 minutes.<br>
 +
6) Add 200 ul LB medium and recover the cells by shaking at 37 °C.<br>
 +
                Ampicillin: 15-30 minutes<br>
 +
                Chloramphenicol: 60-120 minutes<br>
 +
7) Plate out the cells on selective LB (10ul)<br>
 +
8) Incubate at 37 °C. Transformants should appear within 12 hrs.<br>
 +
<br>
 +
<br>
 +
<b>Result</b>:  BBa_J04450: many colonies, BBa_K649304 no colonies, plate rest<br>
<!-- === To here === -->
<!-- === To here === -->
</div>
</div>
</html>
</html>

Latest revision as of 10:46, 23 August 2013

Phage Sensor

Wednesday 7th August

Glycerol Stock of sSP008-sSP0011, M13 Test, Single Colonies, Liquid cultures, PCR and Transformation


Glycerol Stock of sSP008-sSP0011
Take 1.5ml of ON culture + 500ul Glycerol (60%)
Freeze at -80°C

M13 Test
Plate from ON culture:
1) 1:1000 - sSP012: sSP009 (plate 200ul)
2) 200 ul sSP012
3) 200ul sSp009

Single Colonies
streak out ON culture of sSP012 to get single colonies

Liquid cultures of pir+ E.coli and pKD13 from Jake

PCR of SPCR4, SPCR7, SPCR8, SPCR9, SPCR10 from circuar backbone, directly taken from Biobrick plate

Protocol

ReagentVolume
1x
Nuclease-free water37,25µL
5x Phusion HF Buffer10µL
10 mM dNTPs1µL
Forward Primer (10 µM)0.5µL
Reverse Primer (10 µM)0.5µL
Template Plasmid0.25µL
Phusion DNA Polymerase0.5µL
Total Volume50µL


Thermocycler Protocol: NEB Phusion
TempTime
Start98°C30 secMelt
Cycle 198°C5 secMelt35 cycles
Cycle 240.5°C / 60°C25 secAnneal
Cycle 372°C30 sec per kbExtend
Finish72°C5 minExtend
Store10°CForeverStore


Transformation Biobricks BBa_K649304 (with pSB1C3 backbone) and BBa_J04450 (with pSB1A3 backbone) into NEB Turbo (heat shock trafo)

1) Thaw competent cells on ice.
2) Place 20 ul of cells in a pre-chilled Eppendorf tube.
3) Add 2.5 ul of plasmid (from Biobrick stock)
4) Mix gently by flicking the tube.
5) Chill on ice for 10 minutes.
4) Heat shock at 42 °C for 30 seconds
5) Return to ice for 2 minutes.
6) Add 200 ul LB medium and recover the cells by shaking at 37 °C.
Ampicillin: 15-30 minutes
Chloramphenicol: 60-120 minutes
7) Plate out the cells on selective LB (10ul)
8) Incubate at 37 °C. Transformants should appear within 12 hrs.


Result: BBa_J04450: many colonies, BBa_K649304 no colonies, plate rest

Retrieved from "http://2013.igem.org/Team:Paris_Bettencourt/Notebook/Phage_Sensor/Wednesday_7th_August.html"