Team:Paris Bettencourt/Notebook/Phage Sensor/Tuesday 30th July.html

From 2013.igem.org

(Difference between revisions)

Latest revision as of 16:56, 22 August 2013

Phage Sensor

Tuesday 30^th July

Liquid culture, Miniprep and Glycerol stock

Liquid culture of:
pCOLA DUET (from liquid culture (29.07.13)) - 20 ml culture
pSSB1 (from liquid culture (29.07.13)) - 5ml culture
pSSB2 (from liquid culture (29.07.13)) - 5 ml culture
pBAC-BA-lacZ (from plate ) - 5 ml culture
pCRISPR (from plate ) - 5 ml culture
pCas9 (from plate ) - 5 ml culture
pCRIPSR::rpsL (from plate ) - 5 ml culture
F+ strain - (from glycerol stock Trojan Horse) - 10 ml culture
Keio Keio delta pyrF (from plate ) - 10 ml culture

Miniprep (pCOLA-DUET = sSP007) using the Thermo Scientific Miniprep Kit

1) Pellet 2x9 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 left over 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°

pSP001= pCOLA-DUET: 65 ng/ul

Glycerol Stock
from overnight culture of MG1655-pCOLA-DUET (sSP007)
Centrifuge 4000 rpm, 10 minutes,
take out liquid
resuspend cells in 0,5 mL glycerol (60%) , 2 mL LB
freeze in -80°C

@@ Line 25: / Line 25: @@
 <b>Miniprep (pCOLA-DUET = sSP007) using the Thermo Scientific Miniprep Kit</b> <br>
   <br>
-) Pellet 2x 9ml of liquid culture (4000rpm, 10 min)<br>
+) Pellet 2x9 ml of liquid culture (4000 rpm, 10 min)<br>
 ) Discard supernatant<br>
-) resuspend the cells in 250ul of resuspension olution<br>
+) resuspend the cells in 250 µL of resuspension solution<br>
-) add 250ul of lysis solution, mix by inverting 4-6 times<br>
+) add 250 µL of lysis solution, mix by inverting 4-6 times<br>
-) add 350ul of neutralization solution<br>
+) add 350 µL of neutralization solution<br>
 ) centrifuge for 5 min<br>
 ) transfer supernatant to spin column<br>
 ) centrifuge for 1 min<br>
 ) discard flow through<br>
-) add 500 ul wash solution and centrifuge for 1 min , discard flow through(repeat this step)<br>
+) add 500 µL wash solution and centrifuge for 1 min , discard flow through(repeat this step)<br>
 ) centrifuge for 1 min to remove left over liquid<br>
-) transfer the column on a 1.5ml tube<br>
+) transfer the column on a 1.5 ml tube<br>
-) add 50ul of elution buffer and incubate for 2 min<br>
+) add 50 µL of elution buffer and incubate for 2 min<br>
 ) centrifuge for 2 min<br>
 ) Nanodrop the concentration and freeze at -20°<br>
   <br>
-pSP001= pCOLA-DUET: 65ng/ul<br>
+pSP001= pCOLA-DUET: 65 ng/ul<br>
 <br>
 <br>
@@ Line 47: / Line 47: @@
 <b>Glycerol Stock</b> <br>
   from overnight culture of MG1655-pCOLA-DUET (sSP007)<br>
-Centrifuge 4000rpm, 10 minutes,<br>
+Centrifuge 4000 rpm, 10 minutes,<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>