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DH5α Incubation in Liquid Media 6/28/2013
- With a pipetter, 3mL of LB+amp was added to a test tube.
- A colony was selected on the culture plate(plate was overgrown), and a circle was drawn where the colony was selected.
- 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.
- 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.
- Using a micropipette, a 2mL centrifuge tube was filled with the incubated liquid media.
- The solution was centrifuged at 13.1g for 3 minutes (If a pellet has not completely condensed centrifuge again until pellet does condense).
- 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)
- Pellet was broken up using the vortex.
- Added 100μL of lysis buffer to the centrifuge tube and inverted 4-6 times and waited 1 min.
- Added 350μL of neutralization buffer. Inverted the solution until it turns yellow, make sure no blue pockets are left and precipitate forms.
- Centrifuged the solution at 16.1g for 2 mins.
- Transfered the solution into a spin column and placed the spin column in a collection tube.
- Centrifuged spin column/collection tube apparatus at 16.1g for 15 seconds.
- The liquid in the collection tube was discarded. (Into the bleach container in the fume hood)
- Spin column was put back into the collection tube, and 200μL of Endo Wash buffer was added to the spin column.
- Centrifuged at 16.1g for 15 seconds.
- 400μL of Zippy Wash buffer was added to the spin column and the apparatus was centrifuged at 16.1g for 30 seconds.
- Spin column was transferred to a new, clean 1.5 mL centrifuge tube.
- 30μL of the Zippy Elution Buffer was added to the column matrix (the white area), and waited 1 min.
- Centrifuged at 16.1g for 15 seconds. (make sure to point the centrifuge tube caps away from the spin direction)
- Discarded spin column, and labeled the centrifuge tube with initials, and plasmid.("PK KAH013")
- Turn on spectrophotometer.
- Open the program "Gen 5 2.0" (make sure spectrophotometer is on FIRST).
- Click "Take 3 Plate Application," and select the "Nucleic Acid Quantification" routine.
- Clean loading plate with Kimtech wipes and DI water.
- 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)
- Mark the blank and the sample properly on the computer, and click "approve."
- After gathering data, clean loading plate with Kimtech wipes and DI water.