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 1: / Line 1: @@
 {{Paris/Calendar_Links|September 2|September 4}}
-=Transformation of yesterday overnight (18 hours) ligation=
+=Construction of rbs-TetR-mRFP-LVA-tripart (L173)=
+[[Image:Part_icon_rbs.png]][[Image:icon_coding.png]][[Image:Part_icon_reporter.png]][[Image:Part_icon_terminator.png]][[Image:Part_icon_terminator.png]]
+==Transformation ==
 Ligation '''L173''':
@@ Line 10: / Line 13: @@
 <br>
 '''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