Team:Paris Bettencourt/Notebook/Trojan Horse/Friday 16th August.html

Trojan Horse

ASDF

16^th 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

1. 1mL MG,F+ + 100ul LB

2. 1mL MG,F+ + 100ul surnageant diluted 1/10

3. 1mL MG,F+ + 100ul surnageant diluted 1/100

4. 1mL MG,F+ + 100ul surnageant diluted 1/1000

5. 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

  1. 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