Team:Paris Bettencourt/Notebook/Phage Sensor/Wednesday 28th August.html


Phage Sensor

Wednesday 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 PCR
pS005 (C1-C5) - pS006 (C1-C7)
1% 100V 20 min

Liquid Cultures (1:100)
sSP001, sSP002, sSP010, sSP011, sSP012, sSP020

Analytical Digest

Plasmid Miniprep 5 ul
H2O12 ul
c10x FastDigest Buffer2ul
FastDigest Enzyme 10,5 ul
FastDigest Enzyme 20,5 ul
Total Volume 20,0 ul
Digest for 15 minutes at 37 °C with shaking

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
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.