Meiosis mapping experiment
Currently, genetic mapping techniques can be used as a way to monitor the means of genomic manipulation, which needs to be realized through the use of molecular genetic techniques. -The four spores within an ascospore are the product of a single meiotic division, and genetic analysis of these tetrads can provide the chaining of genes in the heterozygous state. If the genes linked to the mitophore are known to be present in the hybrid, then it is possible to map the genes associated with their mitophores.
Operation method
Meiosis mapping experiment
Materials and Instruments
Yeast strains Move Day 1 For more product details, please visit Aladdin Scientific website.
Digestive enzymes 100T
Capillary tips Strong glue Optical glass fiber YPD plates YPD culture tubes
Preparation of Microscopic Needles Tetrads can be prepared by hand drawing fine glass filaments or by using optical fiber glass filaments as described in "Techniques and Protocols 23, Preparation of Tetrads". Needle preparation will be demonstrated and you will be provided with some of the necessary items so that you can prepare your own. Needles and fixators are also commercially available.
Treatment with Demethylase The instructor will attach diploid strain 3-3 to a spore-forming medium 3 days prior to the start of this course. Spore-forming cultures will be observed under a microscope and non-spore-forming cells will be identified as well as tetrasporic ascospores, and ascospores with fewer than four spores. Each student will schedule 3 days to prepare 20 dissected tetraspores from 4 live spores and process the cells by the enzyme ablation method. Microscopic manipulation of tetrads is described in "Techniques and Programs 22, Tetrad Dissection."
Day 3
Complete the dissection of the tetrads.
Day 5
Each student must prepare two parent plates with 4 viable spore colonies on them (10 tetrads per plate) using a sterile flat toothpick and template (Appendix D, Plate Scribing Template). Include two parental control strains on each plate.
Incubate overnight at 30°C.
Day 6
Photocopy tetrads on plates to test medium Photocopy master plates onto the following media: ypd, sc-leu, sc-arg, sc-ade, sc-trp, ypd+cyh, and 6 or more ypd plates (see tips below before starting). The first YPD plate is kept as a fresh master plate for later use. The remaining YPD plates are used to mate spore colonies to the test strain. All plates are incubated at 30°C.
Tip: Making 12 photocopies from an original plate is a lot of work. After 6 photocopies, place a new fleece pad on the photocopying mold and make new photocopies with the master plate. Because the master plate is used twice, the first time the fleece pad is used, it is important to make a lighter photocopy so that the cells remain on the original plate for the second photocopy. Check after each photocopy and you should see a slight settling of cells in their corresponding arrangement on the master plate. If you don't see it, try again. Alternative method.
Spore colonies are inoculated into a microtitre plate containing sterile water, and these cells are then transferred to different test plates using a multitip inoculator, known as the froggingtechnique. Cross-feeding can occur between ^J and strains, so it is important to lightly photocopy onto SC-trp plates.
Preparation of mating type and allele test organisms Cultivate test strains 3-4 through 3-13 in 5 ml of YPD medium Alternatively, prepare large plaques of the test strains in YPD medium. incubate overnight at 30°C.
Day 7
Prepare mating test mosses using either method.
a. Centrifuge 5 ml of cultures of strains 3-4 and 3-5 (tabletop centrifuge 2000 r/min for 5 min). Resuspend the organisms in a sterile screw-cap test tube with 4 ml of YPD. Add 0.15 ml sterile water and 0.15 ml resuspended culture of strain 3-4 to two SD plates (remaining cell stock at 4°C). Spread the organisms with a sterile stick and place the plates horizontally to allow the plates to dry. Treat strain 3-5 similarly.
b. Scrape a fresh spot of strain 3-4 from the YPD plate with a collection rod and resuspend in 4 ml of YPD liquid medium. Incubate overnight to supersaturate the cell concentration. Apply 0.2 ml of bacterial solution to two SD plates. Do the same with strain 3-5. after the liquid has evaporated, photocopy the plate onto the moss.
Preparation of Allelic and Complementation Test Mosses The preparation of these mosses was done in the same way as in the "Mating Mosses" except that the MATa and MATa type test strains were placed on the same plate. Prepare 4 types of test mosses and two of each. The arg4 test mosses (strains 3-6 mixed with 3-7 and strains 3-8 mixed with 3-9) were spread on SC-ura-his plates. For Trp Complementary Moss (strains 3-10 mixed with 3-11 and strains 3-12 mixed with 3-13), apply cells to the SC-trp plate. Allow the liquid to dry before photocopying the plate onto the mushroom moss.
Duplicate plates to test mosses were photocopied onto 6 test mosses using YPD photocopies of 6 tetrads prepared the previous day as a source. For each test moss, a new fleece pad was used to transfer the tetrads to the test moss by imprinting the YPD plate onto the fleece pad and then imprinting the test moss plate onto the fleece pad. The test plates were incubated at 30°C overnight.
Day 9
Photocopy the allelic test moss from the SC-um-his plate onto the SC-arg plate and refrigerate. At the end of the day, the plates are collected by a laboratory assistant and irradiated with a STRATAGENE Stmtalmker 7500|uJ UV lamp (254nm).
The growth of mating type test plates and Trp complementary plates was analyzed. Since tr and defective strains can be symbiotic with defective strains, it was necessary to photocopy the SC-trp plate onto a new SC-trp plate and analyze it the next day.
Day 10/11
Analyze the arg4 Allele Test Plate and the Trp Complementary Plate.
Day 11
Determine the number of PD, NPD, and T-type tetrads that segregate after each pair of markers in the hybridization. Record the PD/NPD/T type information on the recording sheet at the end of this experiment. In addition, the frequency of second split segregation for each marker was recorded. The distance between each gene and the mitophore was determined by searching for information about each gene map in the Yeast Genome Database (SGD; http://www.pathway,yeastgenome.org/). Calculations were made using the collected information:
1) the mapping distance between each analyzed gene and all other analyzed genes 2) the distance between each gene and its interphase (how does this compare to the information provided by SGD?) 3) Gene conversion frequencies (3:1 or 1:3 segregation) for all the alleles we studied Day 14 Preparation for Submission