Protocol. α4-200™ plasmid purification kit

Prior to using the kit for the first time, add 8 mL of 96% ethanol to the bottle labeled “Solution #3”. Store the kit in the refrigerator. 1. Collect cells from 1.5 mL of overnight E. coli cell culture by centrifugation in micro-centrifuge (13 rsf) for 2 minutes and remove supernatant (by opening and inverting the tube, then giving it 1 – 2 intense shakes down into a waste receptacle). 2. Re-suspend cells in the remaining (~30 µl) supernatant using a Vortex. 3. Add 135 µl of Solution #1 per tube. 4. Thoroughly mix the contents of tubes (shaking tubes 2 – 3 times should suffice), and place the tubes at 37oC for 5 – 15 minutes. 5. Transfer tubes to 80oC and continue incubation for an additional 15 minutes. 6. Centrifuge tubes for 10 – 25 minutes at 13 rsf. 7. Remove pellets from tubes by inserting a sterile toothpick (provided in the kit) into each pellet, spinning it inside of pellet, and pulling the toothpick out of the tube together with the stuck pellet. Note: We recommend using metal forceps sterilized by flame for taking toothpicks out of the storage container. 8. Add 145 µl of Solution #2 per tube to the remaining supernatant and mix the contents by gently inverting the tubes several times (for plasmids larger than 20 k.b.) or by intensely shaking 2 – 3 times. 9. Centrifuge tubes for 10 minutes at 13 rsf. 10. Remove supernatant by aspiration. Note: We recommend using faucet aspirator/vacuum pump). 11. Add 50 µl of Solution #3 (remember to add ethanol) per tube. 12. Centrifuge tubes for 2 minutes at 13 rsf. 13. Remove supernatant by aspiration. 14. Add 50 µl of Solution #4. 15. Incubate at 37oC for 5 – 10 minutes. 16. The purified DNA is ready for further manipulations! This is another page that is linked to the Chapter 10 via the Chapter number used and is sorted based on the TOC Order.

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