Team:Paris Bettencourt/Notebook/Phage Sensor/Saturday 13th July.html

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PCR circular and linear, Conjugation of XL-10 (with sSP011), Patches, Digestion M13 backbone, RFP insert, Gel of PCR
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<TR><TD><b>Thermocycler Protocol: NEB Phusion</b></TD><TD> </TD><TD> </TD><TD> </TD><TD> </TD></TR>
<TR><TD><b>Thermocycler Protocol: NEB Phusion</b></TD><TD> </TD><TD> </TD><TD> </TD><TD> </TD></TR>
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<TR><TD> </TD><TD>Temp</TD><TD>Time</TD><TD> </TD><TD> </TD></TR>
<TR><TD> </TD><TD>Temp</TD><TD>Time</TD><TD> </TD><TD> </TD></TR>
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<TR><TD>Start</TD><TD>98°C</TD><TD>30 sec</TD><TD>Melt</TD><TD> </TD></TR>
<TR><TD>Start</TD><TD>98°C</TD><TD>30 sec</TD><TD>Melt</TD><TD> </TD></TR>
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<TR><TD> </TD><TD> </TD><TD> </TD><TD> </TD><TD> </TD></TR>
<TR><TD> </TD><TD> </TD><TD> </TD><TD> </TD><TD> </TD></TR>
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<TR><TD>Cycle 1</TD><TD>98°C</TD><TD>5 sec</TD><TD>Melt</TD><TD> 35 cycles </TD></TR>
<TR><TD>Cycle 1</TD><TD>98°C</TD><TD>5 sec</TD><TD>Melt</TD><TD> 35 cycles </TD></TR>
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<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 2</TD><TD>40.5°C / 60°C </TD><TD>25 sec</TD><TD>Anneal</TD><TD></TD></TR>
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<TR><TD>Cycle 3</TD><TD>72°C</TD><TD>5 min</TD><TD>Extend</TD><TD> </TD></TR>
<TR><TD>Cycle 3</TD><TD>72°C</TD><TD>5 min</TD><TD>Extend</TD><TD> </TD></TR>
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<TR><TD> </TD><TD> </TD><TD> </TD><TD> </TD><TD> </TD></TR>
<TR><TD> </TD><TD> </TD><TD> </TD><TD> </TD><TD> </TD></TR>
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<TR><TD>Finish</TD><TD>72°C</TD><TD>5 min</TD><TD>Extend</TD><TD> </TD></TR>
<TR><TD>Finish</TD><TD>72°C</TD><TD>5 min</TD><TD>Extend</TD><TD> </TD></TR>
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<TR><TD>Store</TD><TD>10°C</TD><TD>Forever</TD><TD>Store</TD><TD> </TD></TR>
<TR><TD>Store</TD><TD>10°C</TD><TD>Forever</TD><TD>Store</TD><TD> </TD></TR>
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Latest revision as of 15:21, 23 August 2013

Phage Sensor

Saturday 13th August

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, pSB1C3

ReagentVolume
1x
Nuclease-free water37.25 ul
5x Phusion HF Buffer10 ul
10 mM dNTPs1 ul
Forward Primer (10 uM)0.5 ul
Reverse Primer (10 uM)0.5 ul
Template Plasmid0.25 ul
Phusion DNA Polymerase0.5 ul
Total Volume50 ul


Thermocycler Protocol: NEB Phusion
TempTime
Start98°C30 secMelt
Cycle 198°C5 secMelt 35 cycles
Cycle 240.5°C / 60°C 25 secAnneal
Cycle 372°C5 minExtend
Finish72°C5 minExtend
Store10°CForeverStore


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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