Team:Paris Bettencourt/Notebook/Phage Sensor/Wednesday 31st July.html

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2)  Wait for OD to reach O,2<br>
2)  Wait for OD to reach O,2<br>
3)  Prepare 4 tubes (in BD tubes) :<br>
3)  Prepare 4 tubes (in BD tubes) :<br>
-
-      Tube 1 = 0,5mL LB with Strain 1 (sSP001) ,5mL LB = control<br>
+
-      Tube 1 = 0,5 mL LB with Strain 1 (sSP001) ,5 mL LB = control<br>
-
-      Tube 2 = 0,5mL LB with Strain 2 (sP001) + 0,5mL LB = control<br>
+
-      Tube 2 = 0,5 mL LB with Strain 2 (sP001) + 0,5 mL LB = control<br>
-
-      Tube 3 = 0,5mL LB with Strain 3 (keio Delta pyrF) + 0,5mL LB = control<br>
+
-      Tube 3 = 0,5 mL LB with Strain 3 (keio Delta pyrF) + 0,5 mL LB = control<br>
-
-      Tube 4 = 0,5mL LB with Strain 4 (XL1) + 0,5mL LB = control<br>
+
-      Tube 4 = 0,5 mL LB with Strain 4 (XL1) + 0,5 mL LB = control<br>
-
-      Tube 5 = 0,5mL LB with Strain 1 (sSP001) + 0,5mL LB with Strain 4 (XL1)<br>
+
-      Tube 5 = 0,5 mL LB with Strain 1 (sSP001) + 0,5 mL LB with Strain 4 (XL1)<br>
-
-      Tube 6 = 0,5mL LB with Strain 2 (sSP002) + 0,5mL LB with Strain 4 (XL1)<br>
+
-      Tube 6 = 0,5 mL LB with Strain 2 (sSP002) + 0,5 mL LB with Strain 4 (XL1)<br>
-
-      Tube 7 = 0,5mL LB with Strain 3 (keio Delta pyrF) + 0,5mL LB with Strain 4 (XL1)<br>
+
-      Tube 7 = 0,5 mL LB with Strain 3 (keio Delta pyrF) + 0,5 mL LB with Strain 4 (XL1)<br>
4)  Incubate 2 hours at 37°C (actually not in the shaker, but we accidently kept them in the shaker...)<br>
4)  Incubate 2 hours at 37°C (actually not in the shaker, but we accidently kept them in the shaker...)<br>
-
5)  Plate 10ul for controls, 100uL, 10ul  for mixed tubes on LB antiobiotics (Tube 5: Chl + Tet, Tube 6: Chl + Tet, Tube 7: Kan + Tet)<br>
+
5)  Plate 10ul for controls, 100uL, 10ul  for mixed tubes on LB antibiotics (Tube 5: Chl + Tet, Tube 6: Chl + Tet, Tube 7: Kan + Tet)<br>
6)  Incubate overnight at 37°C<br>
6)  Incubate overnight at 37°C<br>
<br>
<br>
<b>Miniprep addgene plasmids</b><br>
<b>Miniprep addgene plasmids</b><br>
-
1) Pellet  4ml of liquid culture (4000rpm, 10 min)<br>
+
1) Pellet  4 ml of liquid culture (4000 rpm, 10 min)<br>
2) Discard supernatant<br>
2) Discard supernatant<br>
-
3) resuspend the cells in 250ul of resuspension olution<br>
+
3) resuspend the cells in 250 µL of resuspension solution<br>
-
4) add 250ul of lysis solution, mix by inverting 4-6 times<br>
+
4) add 250 µL of lysis solution, mix by inverting 4-6 times<br>
-
5) add 350ul of neutralization solution<br>
+
5) add 350 µL of neutralization solution<br>
4) centrifuge for 5 min<br>
4) centrifuge for 5 min<br>
5) transfer supernatant to spin column<br>
5) transfer supernatant to spin column<br>
6) centrifuge for 1 min<br>
6) centrifuge for 1 min<br>
7) discard flow through<br>
7) discard flow through<br>
-
8) add 500 ul wash solution and centrifuge for 1 min , discard flow through(repeat this step)<br>
+
8) add 500 µL wash solution and centrifuge for 1 min , discard flow through(repeat this step)<br>
-
9) centrifuge for 1 min to remove left over liquid<br>
+
9) centrifuge for 1 min to remove leftover liquid<br>
-
10) transfer the column on a 1.5ml tube<br>
+
10) transfer the column on a 1.5 ml tube<br>
-
11) add 50ul of elution buffer and incubate for 2 min<br>
+
11) add 50 µL of elution buffer and incubate for 2 min<br>
12) centrifuge for 2 min<br>
12) centrifuge for 2 min<br>
13) Nanodrop the concentration and freeze at -20°<br>
13) Nanodrop the concentration and freeze at -20°<br>
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<b>Glycerol Stock of addgene plasmids</b><br> (see Database for strain reference)<br>
<b>Glycerol Stock of addgene plasmids</b><br> (see Database for strain reference)<br>
  from overnight culture<br>
  from overnight culture<br>
-
Centrifuge 4000rpm, 10 minutes,<br>
+
Centrifuge 4000 rpm, 10 minutes,<br>
take out liquid<br>
take out liquid<br>
-
resuspend cells in 0,5mL glycerol (60%) , 2mL LB<br>
+
resuspend cells in 0,5 mL glycerol (60%) , 2 mL LB<br>
freeze in -80°C<br>
freeze in -80°C<br>

Latest revision as of 17:01, 22 August 2013

Phage Sensor

Wednesday 31st July

Conjugation, Miniprep addgene plasmids,Glycerol Stock of addgene plasmids



Conjugation

sSP001, SP002, keio Delta PyrF
F+ comes from XL1 Blue (glycerol stock Trojan Horse) => F+ is tetR

Protocol :
1) From O/N cultures Dilute strains 1/100 in LB
2) Wait for OD to reach O,2
3) Prepare 4 tubes (in BD tubes) :
- Tube 1 = 0,5 mL LB with Strain 1 (sSP001) ,5 mL LB = control
- Tube 2 = 0,5 mL LB with Strain 2 (sP001) + 0,5 mL LB = control
- Tube 3 = 0,5 mL LB with Strain 3 (keio Delta pyrF) + 0,5 mL LB = control
- Tube 4 = 0,5 mL LB with Strain 4 (XL1) + 0,5 mL LB = control
- Tube 5 = 0,5 mL LB with Strain 1 (sSP001) + 0,5 mL LB with Strain 4 (XL1)
- Tube 6 = 0,5 mL LB with Strain 2 (sSP002) + 0,5 mL LB with Strain 4 (XL1)
- Tube 7 = 0,5 mL LB with Strain 3 (keio Delta pyrF) + 0,5 mL LB with Strain 4 (XL1)
4) Incubate 2 hours at 37°C (actually not in the shaker, but we accidently kept them in the shaker...)
5) Plate 10ul for controls, 100uL, 10ul for mixed tubes on LB antibiotics (Tube 5: Chl + Tet, Tube 6: Chl + Tet, Tube 7: Kan + Tet)
6) Incubate overnight at 37°C

Miniprep addgene plasmids
1) Pellet 4 ml of liquid culture (4000 rpm, 10 min)
2) Discard supernatant
3) resuspend the cells in 250 µL of resuspension solution
4) add 250 µL of lysis solution, mix by inverting 4-6 times
5) add 350 µL of neutralization solution
4) centrifuge for 5 min
5) transfer supernatant to spin column
6) centrifuge for 1 min
7) discard flow through
8) add 500 µL wash solution and centrifuge for 1 min , discard flow through(repeat this step)
9) centrifuge for 1 min to remove leftover liquid
10) transfer the column on a 1.5 ml tube
11) add 50 µL of elution buffer and incubate for 2 min
12) centrifuge for 2 min
13) Nanodrop the concentration and freeze at -20°

See Database for plasmid reference
pCas9:
pCRISPR:
pBac-LacZ:
pCRISPR::rpsL:

Glycerol Stock of addgene plasmids
(see Database for strain reference)
from overnight culture
Centrifuge 4000 rpm, 10 minutes,
take out liquid
resuspend cells in 0,5 mL glycerol (60%) , 2 mL LB
freeze in -80°C