Freimoser:Protocols:FastYeastTransformation: Difference between revisions
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== Protocol: Fast yeast transformation== | == Protocol: Fast yeast transformation== | ||
#Add 50 | #Add 50 μl carrier DNA to a 1.5 ml tube. | ||
#scrap cells from plate and add to the carrier DNA. | #scrap cells from plate and add to the carrier DNA. | ||
#Add in the following order: | #Add in the following order: | ||
##1-2 | ##1-2 μl DNA-plasmid (up to 30-50 ul) | ||
##240 | ##240 vl PEG (50%) | ||
##36 | ##36 μl Li-Ac (1M) | ||
#resuspend with blue tip | #resuspend with blue tip | ||
#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 | #spin down, resuspend in 100μl H<sub>2</sub>O and plate everything on corresponding medium | ||
== Material: == | == Material: == | ||
*Plasmid: 100 ng - 1 | *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). | *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 | *50% poly-ethylen-glycol (PEG) (MW 33-50) solution, filter-sterilized |
Revision as of 06:52, 31 October 2006
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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 vl 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 100μl 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