Freimoser:Protocols:FastYeastTransformation
From OpenWetWare
(Difference between revisions)
| Line 22: | Line 22: | ||
#incubate at 30°C or RT at least 30 min | #incubate at 30°C or RT at least 30 min | ||
#heat shock: 45°C, 15 min (or 42°C for 20 min) | #heat shock: 45°C, 15 min (or 42°C for 20 min) | ||
| - | #spin down, resuspend in 100ul | + | #spin down, resuspend in 100ul H<sub>2</sub>O and plate everything on corresponding medium |
| + | |||
== Material: == | == Material: == | ||
Revision as of 16:54, 23 October 2006
Protocol: Fast yeast transformation
- Add 50 µl carrier DNA to a 1.5 ml tube.
- scrap cells from plate and add to the carrier DNA.
- Add in the following order:
- 1-2 µl DNA-plasmid (up to 30-50 ul)
- 240 µl PEG (50%)
- 36 µl Li-Ac (1M)
- resuspend with blue tip
- incubate at 30°C or RT at least 30 min
- heat shock: 45°C, 15 min (or 42°C for 20 min)
- spin down, resuspend in 100ul H2O and plate everything on corresponding medium
Material:
- Plasmid: 100 ng - 1 µg
- carrier DNA: salmon/herring sperm DNA in H2O, 2mg/ml stock, 500 µl aliquots, heat-inactivated at 95°C for 5-6 min and put on ice before use (has to be done once).
- 50% poly-ethylen-glycol (PEG) (MW 33-50) solution, filter-sterilized
- 1M Li-Acetate, autoclaved
Reference
- Gietz D, St Jean A, Woods RA, and Schiestl RH. . pmid:1561104.
