User:Hetmann/Notebook/2006-11-18

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Harvard-Yale

[The Game] Go Harvard! (- and Dartmouth)

Goals for 11/18

    • Progression
      • Step 1: Midiprep
        1. B0015
        2. J04500+KaiB
        3. J04500+KaiC
        4. J04500+KaiA+J04500+KaiC
      • Step 2: Digestion
        1. B0015/SP important
        2. J04500+KaiB/XP important
        3. J04500+KaiC/XP
        4. J04500+KaiA+J04500+KaiC/SP

Efficient Midiprep Protocol (for HC plasmid)

Pre-Midiprep
  1. Put buffer P3 in the refrigerator.
  2. Check buffer P2 for precipitate; if there is then redissolve @ 37°C.
Midiprep
  1. Harvest the bacterial cells by centrifugation at 6000 x g for 15 min at 4°C.
      • Big centrifuge: SLA-1500; Rotor code 28; 6290 rpm.
      • Big centrifuge: HB6; Rotor code 23; 6060 rpm.
    • Alternatively, centrifuge cultures in 50 mL falcon tubes for 15 minutes at 4400 rmp in room 5096, and dump supernatant. Simultaneously, turn on the big centrifuge in room 5088, fill a bucket of ice, and place the medium rotor into the big centrifuge.
      • Medium rotor: SS34, rotor code 05.
  2. Resuspend the bacterial pellet in 4 ml Buffer P1.
  3. Add 4 ml Buffer P2, mix by inverting 4-6 times, then incubate at room temperature for 4 minutes.
  4. Add 4 ml Buffer P3, mix by inverting 4-6 times, then incubate on ice for 15 minutes.
  5. Mix the sample again.
  6. Centrifuge at 13,000 rpm for 30 min at 4°C, and remove the supernatant promptly. Concurrently, equilibrate a QIAGEN-tip 100 by applying 4 ml Buffer QBT, and allow the column to empty by gravity flow.
    • Centrifuge in non-glass tube. '<~ this should be all of the tubes present in lab'
  7. Centrifuge the supernatant again at 13,000 x g for 5 min at 4°C, and remove the supernatant promptly.
  8. Apply supernatant to QIAGEN-tip and allow it to enter the resin by gravity flow.
  9. Wash the QIAGEN-tip twice with 10 ml Buffer QC, and allow to move through the QIAGEN-tip by gravity flow.
  10. Elute DNA with 5 ml QF.
  11. Collect the eluate in a 10 ml tube.
  12. Precipitate DNA by adding 3.5 ml room-temperature isopropanol to the eluted DNA.
  13. Mix and centrifuge immediately at 11210 rpm for 30 minutes at 4°C, and carefully decant the supernatant. Then make sure all of the isopropanol is out.
  14. Wash DNA pellet with 2 ml of room-temperature 70% ethanol and centrifuge at 11210 rpm for 10 minutes.
  15. Carefully decant the supernatant, and don't disturb the pellet - nevertheless, make sure that no ethanol remains.
  16. Air-dry the pellet for 5-10 min, and redissolve the DNA in 250 ul ddH2O

Estimated time: 170 minutes/ 2h 50m

Adapted from the QIAGEN Midiprep protocol.

Word(s) of the day: karmic retribution
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