University:
Swansea University
Course:
PM134 | Microbiology
Academic year:

2023
Views:

44

Pages:

2
Author:

Sanjeev K

EXPERIMENT NO:3 DATE: 11-10-2022 RAPID ISOLATION OF YEAST GENOMIC DNA AIM To isolate yeast genomic DNA MATERIALS REQUIRED *YPD Medium * Triton X-100 * SDS Macl *Tris-HC1 EDTA * Phena chloroform * Ethanol * Te Buffa * Centrifuge * Agarose gel electrophoresis system * opendray tubes PROCEDURE 1. Transfer 15ml of liquid culture of Yeast (20-24h grown) at 30°c in YPD into ependroff tube centrifuge at 15,000 apm, 5min Remove supernatant 2. Add 200ml lysis beiffer in to the pellet 3. Immerse tubu in water bath for dry ice ethand fee & min; transfer to 95% I min to thaus quickly Vortex fa Bosee. 4. Add 200 µl phenol chloroform (1:D; Vortex for 2 min and centrifuge ar 15,000 spm for 3min, sroom temperature 5. Transfer upper aqueous phase to another eppendroff, containing 400µl ice cold 100% ethanol Mix gently by Inversion 6. Incubate al room temperature for 5 min Allematively. precipitate al -20°C to increase yield. 7. Centrifuge at 15,000 rpm, 5min, socmtemperature. Remove supernatant with Pasteur pipetle. 8. Wash peller with 0-5 m² (500ml) 70% ethanol, spindgión En as deseri- bed in step7 9. Remove supernatant. Air dry the pellets at room temperature Thoa 5 min 10 Resuspend in 15 50ml 10 lxff (Sample from plate should be resuspended in 10ml buffer] 11 Add Dasul RNay Run the yeast genormie DNA nagarese gel labophoresis. CHEL CASTING * Seal the tray using cellophane. Prepare 30 ml 0.8% agarose sedution using * Beil and dische agandre. Add 3u ft. Br The buffer Cool to 50-60°C Pour into gel tray with comb Alloco it to sclidify * Remove comb and tape Mount the gel in tank filled with TBE buffer Mix 10ml DNA + 2M1 gel loading dye * Load the sample in to wells, electrophoresis at 50V OBSERVATION Orange colour bands were observed RESULTS Genomic DNA of yeast was Bust and Grab' method. succesfully holated by