Team:Paris Bettencourt/Notebook/Phage Sensor/Wednesday 28th August.html
From 2013.igem.org
Revision as of 13:59, 7 September 2013 by Marguerite (Talk | contribs)
Phage Sensor
ASDFWednesday 28th August
Gel Colony PCR, Liquid Cultures (1:100), Analytical Digest, Gel Ligation/Gibson, Gel Analytical digest, DpnI digest of SPCR5, SPCR6, SPCR7, Gel DpnI digest, PCR purification of DpnI digested SPCR5, SPCR6, SPCR7, Stress test microscope and M13 Test.
Gel Colony PCRpS005 (C1-C5) - pS006 (C1-C7)
1% 100V 20 min
Liquid Cultures (1:100)
sSP001, sSP002, sSP010, sSP011, sSP012, sSP020
Analytical Digest
Reagent | Volume |
Plasmid Miniprep | 5 ul |
H2O | 12 ul |
c10x FastDigest Buffer | 2ul |
FastDigest Enzyme 1 | 0,5 ul |
FastDigest Enzyme 2 | 0,5 ul |
Total Volume | 20,0 ul |
Gel Ligation/Gibson
pS005 - pS006 - M13/RFP
1% 100V 20min
Gel Analytical digest
pS005 (C1-C5)-pS006 (C1-C7)
1% 100V 20min
DpnI digest of SPCR5, SPCR6, SPCR7
add 4ul of DpnI into each tube, mix and incubate for 1h at 37°C
Gel DpnI digest
let each 5ul run on a gel
1%, 100V 20min
PCR purification of DpnI digested SPCR5, SPCR6, SPCR7
Add 5 volumes of Buffer PB to 1 volume of the PCR sample and mix.
To bind DNA, apply the sample to the QIAquick column and centrifuge for 30–60 s.
Discard flow-through. Place the QIAquick column back into the same tube
To wash, add 0.75 ml Buffer PE to the QIAquick column and centrifuge for 30–60 s.
Discard flow-through and place the QIAquick column back in the same tube.
Centrifuge the column for an additional 1 min.
Place QIAquick column in a clean 1.5 ml microcentrifuge tube.
To elute DNA, add 30 μl Buffer EB to the center of the QIAquick membrane and centrifuge the column for 1 min.
Stress test microscope
Dilute ON cultures of sSP001, sSP002, sSP010, sSP011, sSP012 1:100 and incubate up to an OD of 0.3
Take out 2ml of sSP001, sSP002, sSP010, sSP011
add:
sSP001: 10ug/ml MMC
sSP002: 10ug/ml MMC
sSP010: 20ul sSP012 supernatant
sSP011: 20ul sSP012 supernatant
incubate for 90 min
take pictures under the microscope (trans and YFP) of (un-)treated samples
M13 Test
Liquid cultures of sSP012 and sSP020
1) 100 μ l of plating bacteria per tube (OD 0.6)
2) Prepare tenfold serial dilutions (10-6 to 10-9 ) of the bacteriophage stock in LB
3) Put 10 μ l/ 100 μ l of each dilution to the bacteria
4) Mix the bacteriophage particles with the bacterial culture by vortexing gently.
5) Add 40 μ l of X-gal solution (working conc) and IPTG (working conc) solution to each of the tubes
6) Add 3ml of Agar to each tube and gently vortexing for 3 seconds
7) Pour the mixture onto plates containing LB medium supplemented with 5 mM MgCl2 and 2x IPTG and Xgal
8) Swirl the plate gently to ensure an even distribution of bacteria and agar.
9) Incubate them at 37°C.