Download EasySelect™ Pichia Expression Kit

Transformation into E. coli
Introduction
Important
Ligation mixtures may be transformed into E. coli and selected on Low Salt LB
medium (see below) with Zeocin™. Transformants are isolated and analyzed for
the presence and orientation of insert. There is no blue/white screening for the
presence of insert with pPICZ or pPICZα. After obtaining the desired
recombinant plasmid, you will be ready to transform into Pichia.
For Zeocin™ to be active, the salt concentration of the medium must remain low
(< 90 mM) and the pH must be 7.5. Prepare Low Salt LB broth and plates using
the recipe in the Appendix, page 53.
Failure to lower the salt content of your LB medium will result in non-selection
due to inactivation of the drug.
Transformation
Guidelines are as follows:
•
Transformation may be performed by either electroporation or chemical
methods. Use your preferred method or refer to general molecular biology
references (Ausubel et al., 1994; Sambrook et al., 1989)
•
Add either Low Salt LB or LB medium to the cells after heat shock or
electroporation to allow them to recover.
•
Plate on Low Salt LB medium with 25 μg/ml Zeocin™.
Note: You may also use SOB, 2XYT, or TB medium, but you may have to increase the
concentration of Zeocin™ to 50 μg/ml to compensate for differences in the salt
concentration.
Analyzing
Transformants
•
Incubate overnight at 37°C.
1.
After transformation, plate 10 μl and 100 μl of the transformation mix onto
Low Salt LB plates with 25 μg/ml Zeocin™ (see above) and select Zeocin™resistant colonies.
2.
Pick 10 Zeocin™-resistant transformants and inoculate into 2 ml Low Salt LB
medium with 25 μg/ml Zeocin™. Grow overnight at 37°C with shaking.
3.
Isolate plasmid DNA by miniprep for restriction analysis and sequencing
(see next page).
4.
Be sure to make a glycerol stock of your purified clone for safekeeping.
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