Basic guidelines for SK1 mini-colonies

 

• Standard = 400cells/250uL YPA-VLN (per well)
• Use colonies/patches < 1 week old
• Use 96 well microtiter plates

 

1. Suspend cells  from single colony or patch in 300 uL ddH₂O in 1.5 ml Eppendorf tub
2. Using probe sonicator (Labline 30W ultrasonic processor, 3mm probe) Sonicate 6s, 3x. Check sample in microscope ,If cells are not fully separated, sonicate again.
3. Determine cell concentration, e.g. by counting cells in microscope on hemacytometer.
4. Inoculate a dilution of the cells into an appropriate volume of medium
5. Example:  For 24 wells  with 400 cells / 250 ul / well, you would need 9600 cells in 6 mL VLN.
6. When determining how many wells are needed for a given experiment, as a general rule, have many extra wells. 
7. Using a repeat pipettor  (e.g. Eppendorf) and sterile  tip, add 250 uL to each well.
8. Wrap edge microtiter plate completely with Parafilm 2X to seal tightly.
9. Grow at 24°C  in incubator.  Be careful not to disturb the plates during incubation.  For example, if different time points are required, use a separate microtiter plate for each day.

 

VLN-YPA (liquid)

 

10g Potassium Acetate

5g  Peptone

2.5g Yeast Extract

                pH 5.8 with Acetic Acid

                to 1L with ddH2O

                Autoclave

 

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