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

2023
Views:

255

Pages:

2
Author:

Sanjeev K

EXPERIMENT NO:4 DATE: 31-10-2022 ISOLATION OF PLASMID DNA AIM Alkaline lyn's method Small male plased preparation from Ecole MATERIALS REQUIRED $LB broth * Amphiulur *O2N NACH * Gluwse * This HCI EDTA 5M Sodium cvetate pH 4.8 & Ethanol *1% 505 RNAGE H Distilled water. * Eppendorf tubes af centrifuge PROCEDURE 1. Transfer 2 me & coth culture to eppendorf tube, centrifuge at 300 rpm foo Imin. Remove super nateunt 2. collect the peller, add 100M GTE (Solution) keep it in ice for 2 min 3. Immediately add 20041 of fresncy prepared Naon WSDS (Solution) y gently tap the tubes. 4. Incubate the tunes for amin, add 15041 & scelicom/potassium auct are solution (solution 11) al mix gently 5. Keep the tubes at room temp for max 5 min and centufuge at 5000 rpm for amin. 6. Biscard the pellel of carefully transfer be supermatant to • fresh ependorf tube 7. Add & velunes of ice cold ethanot to the tube and Incubate for 20 min at do°C 8. After incubation centrifuge 14,000 rpm for 15 min 9. wash pellet in ime To% ethanol of centrifuge at 5000 apm for smin 10. Dry the pellet at room temperature to remove etiamol 10 Resuspend in 25 50ml 16 xyfer (Sampler from plate should be resuspended in 10ml buffer] 11 Add 0-2541 RNay Run the gel datrophoresis yean genorme DNA n agaron CHEL CASTING * Seal the tray using cellophane. Prepare 30 ml 0.8% agarose slution wing. TBE buffer * Beil and dissolve agarose. Add 3,41 Et Br. * Cool to 50-60°c Pour into gel tray win comb Allove it to * solidify Remove comb and tape Mount the get in tank filled wit TBE buffer Mix 10ml DNA + 2M1 gel loading dye * Load the sample in to wells, electrophoresis at 50V OBSERVATION Orange dour bands were observed RESULTS Genomic DNA of yeast was succesfully holated by Bust and Grab' method.