Haynes Lab:Notebook/Lab Traning - Pradyumna Kadambi/2013/06/28

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DH5α Incubation in Liquid Media 6/28/2013

  1. With a pipetter, 3mL of LB+amp was added to a test tube.
  2. A colony was selected on the culture plate(plate was overgrown), and a circle was drawn where the colony was selected.
  3. Micropipette tip was lightly touched to the colony making sure not to scrape any of the agar gel. Micropipette tip was then ejected into test tube.
  4. Test tube was incubated with shaker for 7 hours at 37 degrees.

DH5α Mini Prep with KAH013 6/28/2013

  • Colonies that Rene had incubated in liquid media on 6/27 were used.
  1. Using a micropipette, a 2mL centrifuge tube was filled with the incubated liquid media.
  2. The solution was centrifuged at 13.1g for 3 minutes (If a pellet has not completely condensed centrifuge again until pellet does condense).
  3. Using a micropipette, the supernatant was removed until 600μL of solution remained in the centrifuge tube. (Discard supernatant into the bleach container in the fume hood)
  4. Pellet was broken up using the vortex.
  5. Added 100μL of lysis buffer to the centrifuge tube and inverted 4-6 times and waited 1 min.
  6. Added 350μL of neutralization buffer. Inverted the solution until it turns yellow, make sure no blue pockets are left and precipitate forms.
  7. Centrifuged the solution at 16.1g for 2 mins.
  8. Transfered the solution into a spin column and placed the spin column in a collection tube.
  9. Centrifuged spin column/collection tube apparatus at 16.1g for 15 seconds.
  10. The liquid in the collection tube was discarded. (Into the bleach container in the fume hood)
  11. Spin column was put back into the collection tube, and 200μL of Endo Wash buffer was added to the spin column.
  12. Centrifuged at 16.1g for 15 seconds.
  13. 400μL of Zippy Wash buffer was added to the spin column and the apparatus was centrifuged at 16.1g for 30 seconds.
  14. Spin column was transferred to a new, clean 1.5 mL centrifuge tube.
  15. 30μL of the Zippy Elution Buffer was added to the column matrix (the white area), and waited 1 min.
  16. Centrifuged at 16.1g for 15 seconds. (make sure to point the centrifuge tube caps away from the spin direction)
  17. Discarded spin column, and labeled the centrifuge tube with initials, and plasmid.("PK KAH013")

Spectrophotometer analysis

  1. Turn on spectrophotometer.
  2. Open the program "Gen 5 2.0" (make sure spectrophotometer is on FIRST).
  3. Click "Take 3 Plate Application," and select the "Nucleic Acid Quantification" routine.
  4. Clean loading plate with Kimtech wipes and DI water.
  5. First, load a 2μL blank of Zippy Elution buffer, then load the sample. (Plate is magnetic, so remember to hold it down when closing the loading plate)
  6. Mark the blank and the sample properly on the computer, and click "approve."
  7. After gathering data, clean loading plate with Kimtech wipes and DI water.

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