I did miniprep for a transformed bacteria based on the protocol, like:
1. Transfer 1 ml culture tube ino the epi
2. Centrifuge at 3500 rpm for 10min
3. Add 250 µl Buffer MP1 (resuspend buffer) and resuspend the pellet so that no cell clumps remain
4. Add 250 µl Buffer MP2 (lysis buffer) and mix gently by inverting the tubes 5 times
5. Incubate at RT for up to 5 min, invert a few times during incubation
6. Add 250 µl Buffer MP3 (Neutralization buffer) and mix gently by inverting the tubes 5 times, incubate for 15min on the ice
7. Clarify the lysate by centrifuging 15 min at 13000rpm
8. Transfer the cleared supernatant (~800 µl) to a fresh 1.5 ml Eppendorf; avoid the flaky white stuff!
9. Add 575 µl RT 100% isopropanol and vortex vigorously
10. Incubate 20 min at -20°C
11. Pellet DNA 10 min at 14000g on 4°C, discard supernatant
12. Wash with ice-cold 70% EtOH, vortex briefly, centrifuge 5 min at14000g, discard supernatant, repeat once
13. Remove as much supernatant as possible by pipetting and air-dry for 15 min
14. Add 50-100 µl ddH2O and incubate 30-60 min at 65°C with ample agitation.
The problem is in the last step. I couldn't dissolve the pellet properly. So, I had to centrifuge it again and collect the supernatant and discard the pellet. I wanted to send the plasmid for sequencing. Do you think this sample is good enough for sequencing?? What can I do to resolve this issue?
Usually there's no problem for me. And I believe the correct term would resuspend in the ddH20. Try to use micropipette and gently resuspend by pipetting.
Thanks for your response. Normally, how long does it take to become dissolved?
The DNA pellet should dissolve almost immediately since nucleic acids are hydrophilic in nature. You might be facing difficulty in resuspending the pellet if traces of ethanol is left in your pellet but since you are air-drying the pellet for 15mins I can't understand how that could happen. Your protocol looks fine could be something with handling.
- Badges
- Science method