Team:HKUST/Protocols/Cell lysis

Cell lysis

Purpose: to lyse E.coli cell to get linearized ligated plasmid.

Materials: cultured transformed E.coli colony, phenol, chloroform, ddH2O

Procedure:

a.Add 40μL ddH2O in a 1.5mL microcentrigfuge tube.
b.Use a toothpick to pick a colony and dissolve it in the water.
c.Add 20μL phenol and 20μL chloroform.
d.Scrape the tube to mix the solution. A well mixed white solution can be seen after this step.
e.Centrifuge it for 10mins (13,000 rpm).
f.Place the supernatant in another tube.
g.Load 10μL to test the result by gel electrophoresis.

Tips:

Ignite the fire for sterile environment when transforming the colony.

Safety tips:

1. Phenol is a strong toxin, make sure to wear gloves and be careful with this chemical.
2. Be careful with the fire, and extinguish it after use.

• Agarose gel preparation and gel electrophoresis
• PCR (Taq, Vent)
• PCR product clean-up
• Enzyme digestion (vector, insert)
• Plasmid DNA extraction
• Gel cutting
• Gel extraction
• Ethanol precipitation
• Ligation
• Transformation to E.coli
• Transformation to yeast
• Cell lysis
• Yeast genomic DNA extraction
• Western blotting
• GFP testing