Latest revision as of 18:53, 16 September 2008

Construction of rbs-TetR-mRFP-LVA-tripart (L173)

Ligation L173:

Insert:vector ratio : 3:1 or 4:1

Transformation of E. coli DH5 alpha competent cells

Defroze of the cells on ice
Add 5 µL of DNA in 100 µL of competent cells (ligation products or pUC19 for positive control)
30 min on ice
Heat schock 45sec at 42°C
2 min on ice
Add 900 µL of SOC
Incubate 1h at 37°C
Centrifugate 5 min at 6000 rpm
Remove 800 µL
Plate the 200 µL left on LB + amplicilline
incubate O/N at 37°C

@@ Line 13: / Line 13: @@
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 '''Transformation of E. coli DH5 alpha competent cells'''
-*100 µL of competent cells
+* Defroze of the cells on ice
-*5 µL of DNA (ligation products or pUC19 for positive control)
+* Add 5 µL of DNA in 100 µL of competent cells (ligation products or pUC19 for positive control)
-*30 min on ice
+* 30 min on ice
-*heat shock 45 s at 42°C
+* Heat schock 45sec at 42°C
-*2 min on ice
+* 2 min on ice
-*0,9 mL SOC
+* Add 900 µL of SOC
-*1 hour at 37°C
+* Incubate 1h at 37°C
-*plating on LB + ampicilline
+* Centrifugate 5 min at 6000 rpm
-*overnight incubation at 37°C
+* Remove 800 µL
+* Plate the 200 µL left on LB + amplicilline
+* incubate O/N at 37°C