Replica Plating

— Ryla Cantergiani 2020/03/30 16:28

Replica Plating is generally, a process of stamping a replica of a set of colonies, spots, or streaks from one agar petri dish (“plate”) to another, usually from a general growth media onto one or more marker or selective media.

In our lab replica plating is used to analyze the results of Tetrad Dissection. Typically the colonies formed by the tetrads on YPD plates are replica plated onto one or more of -Met and -Lys (the markers for the two mating types in our yeast deletion sets), G418 (kanamycin resistance, the deletion cassette used in our deletion collections), and A and alpha tester plates to be re-replica plated into minimal media. This protocol addresses these particular plates. If we are running low or are out of these plates, you must make some more or tell Olivia.

1. After your dissected tetrads have been in the 30°C Incubator for a couple of days, you can replica plate. You'll need Replica Plater, Velvets, selective plates.
2. The replica plater and velvets are in DRAWER ???. The selective plates are in the large 4°C fridge, on the second from the bottom shelf.
3. Make mating type testers
   1. Make tester strain lawns for your A and alpha tester plates. This is done by taking 150μL (per YPD plate needed) dH2O in 1.5 ml Eppendorf tubes and twirling in it a pipette tip with tester strain yeast on it (the tester strain which is itself alpha, which tests for A, is JO297 and the one which is itself A, which tests for alpha, is JO296; you should have a YPD plate of these two sitting around for just this purpose, and freeze a stock of each the first time you use them- see Freezing Strains). Flame sterilize and ethanol glass lawn spreader, then gently touch a part of the YPD plate with no yeast on it to cool the surface a little before actually contacting the yeast. Pipette 150 μL onto plate and spread lawn until you feel resistance. Let dry before plating.
4. Starting with a clean velvet, gently place your tetrad plate onto the velvet, tapping lightly with your fingertips to make an impression. Remove the tetrad place, and keeping the same orientation, transfer to a fresh YPD, G418, C-Met, C-Lys, C-Ura, and finally a tester plate which you spread with A TESTER STRAIN JO296 / AM22? (label this “w/ A=alpha”).
5. Using a second velvet, make a new impression from your YPD plate and transfer to a tester plate which you spread with ALPHA TESTER STRAIN JO297 / AM227 (label this “w/alpha=A”). You may want to end all replica platings with a clean YPD plate as a control to show that cells carry over through all replicas.
6. Place plates, including YPD original, into the 30°C Incubator for a couple of days, then photograph, score, and store in the 4°C fridge if needed. You will then RE-REPLICA your A and alpha tester strain plates to minimal media “B” plates, wait two additional days at 30°C, and score for mating types. You may then proceed to freezing down desired strains, and PCR verification of strains by Colony PCR.

DO NOT KEEP RESTREAKING YOUR TESTER STRAINS FROM PLATE TO PLATE TO “SAVE” YOU TIME REPULLING THEM. The strains can pick up suppressors, which an entirely other issue to deal with, so just repull them from the freezer as needed.

Velveteen Squares: You can use any VELVETEEN from a fabric store (bring an old velvet for comparison, do not buy fabrics other than VELVETEEN), or use VWR P/N 89033-116

list the full manufacturer name and manufacturer part number for all reagents, and then for all equipment. If these already have wiki pages you should be able to get the numbers there. PART NUMBERS will always have two sections, first Reagents and then Equipment. Please place these items in 2 tables.