Haynes Lab:Notebook:Shared/Cloning Bootcamp/2013/06/28

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Jan Simper - 06/28/14

  • Learned about bacterial growth in the agar plates and about "overgrowth," which occurs after the bacteria are left to incubate in the agar plate too long. It is preferred to get the main colonies and not the satellite colonies for the incubation.
  • Pre-miniprep protocol was followed as listed:
  • 1. Circle a colony on the agar plate and using a pipette tip, lightly touch the colony without getting too much of the agar.
  • 2. Eject the tip into a 3 mL tube of LB broth without having the tip touch the walls.
  • 3. Incubate in the shaker at 37 C from 7 hours to overnight.
  • From 3 mL in the incubating tube, we need to get down to 600 ul.
  • 1. As much fluid as possible was poured into a 2 mL centrifuge tube and centrifuged at 13.1 g for 3 minutes. Remember to mark the tube at this point.
  • 2. Using the L1000 pipette, the supernatant was siphoned off in a fume hood until there was 600 mL left in the centrifuge tube. The supernatant should be ejected into the E. coli waste bin in the fume hood. The tube was vortexed to break up the pellet.
  • 3. At this stage, protocol continued because the tubes were not marked when centrifuged in step 2; however, if we were certain about which tubes were which, the remaining fluid from the incubation tube should be poured into the 2 mL centrifuge tube and steps 1-2 repeated to get as much DNA as possible into the 2 mL centrifuge tube. The lack of this step may have contributed to the later results (see below).
  • 4. At this stage, the Zyppy Plasmid Protocol was followed as in this image:

However, the protocol is repeated in writing here.

  • 5. Add 100 μL 7x lysis buffer (blue) to the centrifuge tube with 600 ul of fluid and invert 4-6 times, allowing 1-2 minutes for lysis. Time limit 2 minutes for lysis.
  • 6. Add 350 μL Neutralization Buffer (yellow) and invert until sample becomes yellow (10 times). Neutralization buffer must be refrigerated. Try to make sure that all blue fluid is gone at the end of this step; any remaining blue "pockets" are not neutralized and will pose problems later.
  • 7. Centrifuged for 2 minutes at 13.1 g.
  • 8. Transfer the supernatant into spin column, getting as much as possible in it, but taking care not to go over the marked line on the column. Put the spin column into collection tube. Centrifuged for 15 secs at 13.1 g. Discarded flow through.
  • 9. Added 200 μL of elution buffer (ENDO Wash Buffer). Centrifuged for 15 seconds at 13.1 g.
  • 10. Added 400 μL of Zyppy wash buffer into column. Centrifuged for 30 seconds at 13.1 g.
  • 11. Transferred column into new 1.5 mL tube. Add 30 μL of Zyppy Buffer and let sit for 1 min. Centrifuged for 15 seconds at 13.1 g. It is important to note that water was used to elute the DNA for today, but the elution buffer might be used for future experiments.
  • 12. Go and start the spectrometer machine before selecting the Gen5 2.0 program on the computer.
  • 13. Take out the cartridge and make sure to clean it with the Kimwipes before use.
  • 14. Put 2 ul of the "blank" (water, in this case) in a slot on the cartridge.
  • 15. Put 2 ul of each DNA sample onto a slot in the cartridge. Supporting one hand with the other, slowly eject the sample until a bubble forms on the end of the pipette tip, then touch this bubble to the hole in the cartridge and eject the rest of the DNA.
  • 16. Put the cartridge in the spectrometer and use the program to select the blank and sample holes, and make sure double-stranded DNA is selected.
  • 17. Compare results to expected results of 80-150 ng/ul for a good sample, and 20-80 ng/ul for a poor return yet usable plasmid DNA. If over 200 ng/ul, probable contamination by genomic DNA occurred. If 260/280 value is below 1.8, probable protein contamination occurred.
  • 18. If a good result is received, label the final tube with name, date, part number, and concentration, and place into -20 C freezer.
  • Results:
  • 21-23 ng/ul. Although this means the protocol was probably successful, as seen by the absorption curve increasing sharply and then decreasing, this is a rather low yield. This could have resulted from not all of the incubation fluid being used (see step 3) or contamination at any point. The 260/280 value was approximately 1.5, which means there was some protein contamination.






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