Team:HKUST/Protocols/Transformation to yeast

Transformation to E.coli

Purpose: To transform desired plasmid DNA in to E.coli in order to amplify it.

Materials: E.coli competent cell, desired plasmid DNA, LB media, agar plate with desired selection antibiotic (ampicillin)

Procedure:

a.Perform ligation or any other manipulation of DNA to yield circularized plasmids.
b.Remove bacterial aliquots (usually the DH5 strain of E.coli) from -70°C freezer and thaw on ice for 10-15 min; briefly flick the tube to resuspend the bacterial cells, but minimize any time the cells are not on ice.
c.Add desired amount of plasmid DNA (usually 1-5μL of a standard ligation or 1.0ng of purified plasmid) to the bacteria at the bottom of the tube, mix it with pipette and incubate 5min on ice.
d.Remove the tube from ice and immediately begin heat shock in a water bath at 42°C for 45 sec.
e.After heat shock, immediately add 500μL of room temperature LB media to the bacterial cells, then incubate in a 37 °C shaker incubator for 45mins.
f.Plate 50μL of the bacterial culture to a LB/agar plate with desired selection antibiotic (ampicillin) using a sterile bacterial streaking rod, and allow the plate to absorb the extra liquid for 15 min on the benchtop at room temperature.
g.Invert the plates and place in the 37°C bacterial incubator overnight.

Tips:

A. The competent cell is quite fragile, so make sure that it does not leave the ice before heat shocking.
B. LB is easily contaminated, make sure to sterile it with fire each time when opening or closing it. Add it quickly to minimize the time of leave it open.
C. Ignite the fire for sterile environment when adding LB.

Safety tips:

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