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Team:Paris Bettencourt/Notebook/Trojan Horse/Friday 16th August.html
From 2013.igem.org
Trojan Horse
ASDF16th August
Results from Infectiveness characterization experiments (Vincent)
See file “phage infectivness 15_08.xlsx”
Results
Transformations : (Aude)
Lac : “tapis” on all the plates even the negative control => plates were not done correctly, antibio added to early => do another transformation
Kan : Some colonies on the plates , but also colonies on the negative control => launch few colonies on liquid media (with Kan, Cm)
Phage infectiveness : (Vincent)
Run PCR (Litmus-Gibbson) (Aude)
Small point around 3000-3500bp
Run another gel with large holes to check if it is correct and purify => Not correct, nothing on the new gel
Transformation : (in chemical competent NEB turbo ) Aude Clovis
Rest of ligation product
1) Thaw competent cells on ice.
These can be prepared using the CaCl2 protocol.
2) Add 5 ul of ligation product
4) Mix gently by flicking the tube.
5) Chill on ice for 30 minutes.
4) Heat shock at 42 °C for 30 seconds.
5) Return to ice for 2 minutes.
6) Add 300 ul LB medium and recover the cells by shaking at 37 °C for one hour
7) Plate on selective media
Plate on :
Tube 1 : negative control Xgal /IPTG /Cm ; Kan/Cm
Tube 2 : ligation lacZ-pacy (5uL) Xgal /IPTG /Cm
Tube 3 : ligation Kan-pACY (5uL) Kan Cm
Tube 4 : native plasmid (pACY) (1uL) Xgal /IPTG /Cm ; Kan/Cm
Gel from PCR litmus (Clovis)
1kB ladder / triple puit (3*50uL) from column 2,3,4 of PCR
50V
Infectiveness characterization experiments (Aude)
-the night before: prepare O/N of F+ cells, prepare O/N of phagemid producing cells (in this case : sT007 )
-centrifugate phagemid producing cells
-filter the supernatant 0.45um, stock it at 4°C (it contains the phages)
-dilute 200x MG, F+ from O/N
-wait until OD600 = 0.7
-immediately mix MG and the supernatant in different proportions
here we planned to try 1/100 (vol supernatant/vol cells), 1/1000 and 1/10000
-
1mL MG,F+ + 100ul LB
-
1mL MG,F+ + 100ul surnageant diluted 1/10
-
1mL MG,F+ + 100ul surnageant diluted 1/100
-
1mL MG,F+ + 100ul surnageant diluted 1/1000
-
1mL MG,F+ + 100ul surnageant diluted 1/10000
-incubate 45 minutes at 37°C for the protein to be expressed.
-
Serial dilute the tubes 10^-1, 10^-2, 10^-3, 10^-4, 10^-5
-
4 times 5 tubes with 900uL LB, add 100ul of one previously made tube, mix and transfert 100ul to next tube.
-
Plate on LB (10^-5), Kan( 10^-2), Amp(10^-5), Kan and Amp(10^-2)
Ligation (Vincent)
masse insert = ration (svt 10) * (taille insert / taille vecteur) * masse vecteur (50-100ng)
CHosen Vector mass = 50 ng
lacZ :
Digestion vector : 6,3 ng/ul
Digestion insert : 6,2
masse insert = 10*449/3770*50 = 59 ng
Protocol
Vector : 8 uL
Insert : 10 uL
Buffer : 2 uL
T4 DNA ligase : 0,2 uL
Let incubate at 22°C for 30 min
Kan :
Digestion vector : 7,8 ng/ul
Digestion insert : 12ng/ul
Insert mass = 10*1052/3770*50 = 140 ng
Protocole Ligation
Vector : 7 uL
Insert : 11 uL
Buffer : 2 uL
T4 DNA ligase : 0,2 uL
Transformation : (in chemical competent NEB turbo (made on the …) vincent
1) Thaw competent cells on ice.
These can be prepared using the CaCl2 protocol.
2) Add 5 ul of ligation product
4) Mix gently by flicking the tube.
5) Chill on ice for 30 minutes.
4) Heat shock at 42 °C for 30 seconds.
5) Return to ice for 2 minutes.
6) Add 300 ul LB medium and recover the cells by shaking at 37 °C for one hour
7) Plate on selective media
Plate on :
Tube 1 : negative control Xgal /IPTG /Cm ; Kan/Cm
Tube 2 : ligation lacZ-pacy (10uL) Xgal /IPTG /Cm
Tube 3 : ligation Kan-pACY (10uL) Kan Cm
Tube 4 : native plasmid (pACY) (1uL) Xgal /IPTG /Cm ; Kan/Cm
PCR pUC18 (pT007)
5*50uL
34 H2O
10 Buffer 5x
1 dNTP
2.5 FW iT007
2.5 RV iT008
0.2 pUC18
0.5 phusion
50uL Tot
Tm = 55°C
te = 2min10
no bands
PCR Litmus (Clovis):
Trouble shooting : since the temperature does not help, that it was not the mini prer = but that we see homodimere between the primers => try with less primers
PCR litmus (pT005)
5*50ul
40 H2O
10 Buffer 5x
1 dNTP
0.5 FW iT005
0.5 RV iT006
0.2 litmus
0.5 phusion
50ul Tot
Tm = 64°C
te = 2min10
