Skatebro:Transformation

From OpenWetWare

(Difference between revisions)
Jump to: navigation, search
Current revision (06:09, 9 February 2008) (view source)
(Removing all content from page)
 
Line 1: Line 1:
-
[[Skatebro:Project08]]
 
-
==Tips==
 
-
*Use flat bottom ''plastic'' tubes (glass tubes absorb dna)
 
-
*Vortex before plating
 
-
*Heat shock 50 secs IS important (30 for envitrogen should be good)
 
-
*Plate 20 microL and 200 microL of cells both (or just 200 if not worried about overgrowth)
 
-
 
-
==Steps==
 
-
#Thaw on ice ~50μl cells (however many you will need) in plastic flat bottom tubes
 
-
#Add DNA, pipette gently to mix (keep volume of DNA less than 5% of the cell volume)
 
-
#Incubate on ice for 30 minutes
 
-
#*Note: If you are in a rush, you can shorten this incubation time to 5-10 min.
 
-
#Heat shock cells for 50 seconds at 42°C.
 
-
#Incubate cells on ice for 2 min.
 
-
#Add 300μL of room temperature [[SOC]] (make sure not contaminated!)
 
-
#Incubate for 1 hour at 37°C on shaker.
 
-
#*Note: Can also save some time here by reducing incubation to ~45 min.
 
-
#*Note: Step can be eliminated if plating on Amp plates, but not most other antibiotics
 
-
#Spread 20 & 200 μl onto plates made with appropriate antibiotic.
 
-
#Grow overnight at 37°C.
 

Current revision

Personal tools