Team:Paris Bettencourt/Notebook/Trojan Horse/Thursday 15th August.html

Trojan Horse

ASDF

15^th August

Miniprep (Clovis)

pACY (pT002) : 109ng/uL

litmus (pT005) : 610 ng/Ul

Ligation (Aude, Clovis ,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 …) Aude Clovis

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

PCR (litmus = Backbone) Vincent

8 tubes, gradient 10°C around 64°C , started from a new miniprep

We see the plasmid => too concentrated

PCR (litmus pour la gibbson) Clovis

sTOO5 primer 005 006 protocol phusion (gradient around 64 + or – 5 °C)

Elongaiton time 2min10. Started with the new miniprep diluted.

Infectiveness characterization experiments (Vincent)

-the night before: prepare O/N of F+ cells, prepare O/N of phagemid producing cells (in this case : sT007 )

After checking GFP fluorescence of sT007 and its negative result, we decided to make a new sT007 glycerol stock. We used a colony from the first successful Infectiveness characterization experiments that was both AMP+KAN resistant and expressed GFP.

The following experiment was conducted with this new sT007 (strain MGZ1).

-centrifugate phagemid producing cells

-filter the supernatant 0.45um, stock it at 4°C (it contains the phages)

-This experiment was conducted both on MGZ1(F+) and MG1655(F+)

-dilute 200x MGZ1 and MG1655 from O/N

-wait until OD600 = 0.7

-immediately mix MGZ1 and the supernatant in different proportions

here we planned to try 1/10 (vol supernatant/vol cells)

1. 1mL MGZ1,F+ + 100ul LB

2. 1mL MGZ1,F+ + 100ul surnageant diluted 1/100

-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, 10^-6

  1. 6 tubes with 900uL LB, add 100ul of one previously made tube, mix and transfert 100ul to next tube.

Plate on LB (10^-4 10^-5 10^-6), Kan(10^-1 10^-2 10^-3), Amp(10^-4 10^-5 - 10^-6), Kan and Amp(10^-1 10^-2 - 10^-3)