Replica Plating of Yeast By Frogging

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Tony Rook
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Replica Plating of Yeast By Frogging

Please find the following protocol:

Replica Plating of Yeast By Frogging

Overview:

This protocol describes how to make replicas of yeast plates using a method called "frogging". Frogging refers to the use of a pronged replica plater to transfer cells to multiple plates. In this protocol, the replica plater has 48 prongs arranged in a 6 x 8 grid spaced to fit into the wells of half of a 96-well plate. The cells to be transferred to the plates are suspended in the wells. It is convenient to "frog" when needing to replica the same strain to many different plates to test markers, as when scoring a cross between two yeast strains.

Procedure:

1. Collect and label plates so that, for each strain, there will be an SD Plate deficient in each prototrophic marker individually, an SD plate supplemented with all amino acids as a positive control, an SD plate lacking all amino acid supplements as a negative control, and three YPAD Plates. One of the YPAD plates will be a permanent master and two will be used to replicate to lawns of mating testers to score the MAT locus.

2. Add about a teaspoon of sterile glass beads to each SD Plate to be supplemented with 100X Drop-Out Mix.

3. Add 300 μl of the first Drop-Out solution to its respective plate(s). For each prototrophic marker, prepare an SD Plate with 300 μl of an 100X Drop-Out Mix containing all the required supplements except that marker.

4. Shake the plate(s) to spread the solution.

5. Repeat Steps #3 to #4 for each labeled SD Plate.

6. Dump the beads into a beaker when plates are dry.

7. Organize the plates according to strain.

8. Put a 96-well assay plate and a large glass Petri dish into an UV Crosslinker (Stratagene).

9. Turn on the UV crosslinker and press Start (the display should read 1200 and count down to 0).

10. Add sterile ddH2O to the Petri dish.

11.Use a 12-channel multipipettor to pipet 100 μl of sterile water into the 96-well assay plate. If you are using more than one assay plate, put one back into the UV crosslinker until you are ready for it.

12. Using a sterile toothpick, transfer yeast colonies into their respective wells. Make a chart of each strain's location as a reference. Make sure to include parent strains. Fill half of the wells of the 96-well plate at a time.

13. Add 95% Ethanol to another large glass Petri dish (this dish does not need to be sterile).

14. Dip the 6 x 8 pronged replica plater into the Ethanol and then into the flame of a Bunsen burner.

15. When the Ethanol has burned off and the replica plater has cooled, dip the plater into the assay plate to coat the ends of the prongs with the yeast suspensions, and gently press the replica plater onto the prepared plate to transfer.

16. Make sure to dip the replica plater into Ethanol and flame after each transfer.

17. Repeat Steps #14 to #15 for each prepared plate.

Solutions:

YPAD.........................................................20 g Bacto Peptone
..................................Autoclave and allow solution to cool
..................................................10 g Bacto Yeast Extract
...........................Add 0.1 volume of 20% Glucose Solution
.................................................................890 ml ddH2O
.........................Add 10 ml of a 100X Adenine Supplement
95% (v/v) Ethanol
Sterile Glass Beads, Approx 4 mm diameter glass beads, washed and autoclaved
Drop-Out Mix (100X)*
......................................................2 mg/ml L-Uracil
..............................................20 mg/ml L-Threonine
.........................2 mg/ml L-Histidine HCl Monohydrate
.............................................2 mg/ml L-Arginine HCl
....................................................15 mg/ml L-Valine
...........................2 mg/ml L-Adenine Hemisulfate Salt
..................................................3 mg/ml L-Tyrosine
.....................................................3 mg/ml L-Lysine
...............................................2 mg/ml L-Methionine
.....................................................10 mg/ml Leucine
...........................................5 mg/ml L-Phenylalanine
..............................................2 mg/ml L-Tryptophan
................................................3 mg/ml L-Isoleucine
SD Plates..........................................................20 g/liter Agar
.......6.7 g/liter Yeast Nitrogen Base (w/o Amino Acids)
.....................................................20 g/liter Glucose
................................................Autoclave for 30 min
Add the appropriate sterile 100X Drop-Out Mix when
the solution has partially cooled and before pouring
the plates or top spread 300 μl of 100X Drop-Out Mix
plate after the plates have hardened.
Adenine Supplement (100X)...............2 mg/ml Adenine Sulfate
.....................................Autoclave
20% Glucose Solution.......................20% Sterile (w/v) Glucose

* Make 100X Drop-Out Mixes lacking the appropriate supplements for prototrophies of the strains of interest. For example, if the strain is ade2- his3- ura3- make a 100X Ade- Drop-Out Mix, a 100X His- Drop-Out Mix, and a 100X Ura- Drop-Out Mix. Each of the Drop-Out Mixes would lack the supplement stated in the name of the mix.

Bioreagents and Chemicals:

L-Tryptophan
L-Threonine
Leucine
L-Phenylalanine
L-Methionine
L-Isoleucine
Yeast Nitrogen Base
Bacto Yeast Extract
Bacto-Peptone
L-Valine
L-Histidine HCl Monohydrate
L-Uracil
L-Tyrosine
L-Lysine
L-Adenine, Hemisulfate Salt
L-Arginine HCl
Glucose
Agar
Ethanol

Reference Link:

http://www.bio.com/protocolstools/protocol.jhtml?id=p2052