This procedure begins with preparing an array of a CP one GFP by first growing a line of cells and then pin out the array on a fresh plate. At the same time, prepare a fresh copy of DMA. Next we mate the ACP one GFP array to the DMA and select diploids, which are then sporulated on sporulation media.
Five days later, the Myotic progeny are germinated on LRK media, on which only MAT A haplos will grow. Through two additional rounds of selection, we recover haplo cells carrying both ACP one GFP, and a gene knocked out on can mycin. Finally, we can pick individual strains directly from the plate and observe the mitochondria morphology by confocal microscopy.
Hi, I'm Chap T from the Law Lab from the Department of Cell and Developmental Biology at the Life Science Institute of the University of British Columbia. Hi, I am Jesse Chao, also from the Loan Lab. Today we'll show you a high throughput method for incorporating or GFP markers in various mutant backgrounds in yeast using a robotic system.
We use this procedure in our laboratory to study the morphology of organelles such as D, endoplasmic reticulum and mitochondria. So let's get started. For this protocol, we use two different yeast strains.
A CP one GFP is a strain with a C terminal GFP tagging of ACP one, which is generated by A PCR mediated homologous recombination using plasmid PKT 1 28. The strain background is BY 7 0 4 3. We also use the DMA strain or deletion mutant array, which is an array of about 5, 000 deletion mutants of non-essential genes.
All of these non-essential genes were knocked out on G four 18. The strain background of the array is BY 47 41. To begin grow Alan of ACP one GFP cells by pouring a five milliliter of overnight ACP one GFP culture onto a hiss plate.
Allow the plate to dry sufficiently by a flame or in a biological fume hood. Once dry, incubate the plate at 30 degrees Celsius overnight. After incubation, use a robot to array the A CP one GFP strain into 1, 536 colonies per plate.
At the same time, prepare a fresh copy of the DMA by replica pinning to YPDG four 18 plates. Afterwards, incubate the plates at 30 degrees Celsius overnight after the strains have grown. Made the A CP one GFP strain with the DMA by replica pinning and overlaying the colonies on YPD plates, incubate the plates at 30 degrees Celsius overnight.
After incubation replica plate, the colonies under SD hiss G four 18 plates in order to select deployed cells, incubate the plates at 30 degrees Celsius overnight. In order to begin sporulation and germination first replica plate, the diploids onto YPDG four 18 plates to increase the efficiency of sporulation in the next step. After incubating at 30 degrees Celsius for one day replica plate, the diploids onto enhanced Sporulation media, which contains reduced levels of carbon and nitrogen sources to induce the formation of myotic spores.
Next, incubate the plates at 25 degrees Celsius for a minimum of five days. After incubation germinate the mat, a myotic progeny by replica plaing the colonies onto LRK media. This media will induce the germination of tabloid cells from spores, incubate the plates at 30 degrees Celsius for two days in order to select cells carrying both the GFP allele and the single mutant allele.
Matt A cells are replica plated under lade G four 18 media, which selects for haplo cells that carry the gene deletion, incubate these plates at 30 degrees Celsius overnight. Next replica plate, the mat, a miotic progeny onto L-H-R-K-G four 18 media to select for growth of single mutants. That also harbors ACP one GFP incubate overnight at 30 degrees Celsius.
The plates can then be stored at four degrees Celsius for up to three months. In order to visualize the desired mutants expressing a CP one GFP directly from plate, start by picking the desired colonies from the plates. Grow the cells at 30 degrees Celsius and YPD or SD hiss media to early log phase.
Finally, visualize a mutants using confocal microscopy with the GFP filter set in this confocal image, a mutant and a wild type with the ACP one GFP marker were visualized by confocal microscopy to study the mitochondria morphology. This particular mutant exhibits disrupted mitochondrial phenotype and is therefore a good candidate for further investigation. We have to have shown you how to efficiently incorporate a multiclonal marker, a CP one GFP into the deletion mutant array by using a robotic system.
When doing this procedure, it's important to make the media freshly and allow the place to have sufficient time to dry. If there are too moist condensation will build up increasing the chance of contamination. Also, you should double check that all selection media are made correctly because they are critical to this protocol.
This procedure can also be used for other types of markers. For example, to visualize er, we routinely use the marker air 11 and GSP. So that's it.
Thanks for watching and good luck with your experiments.
