Making K. Rhaeticus stock from Cryostock
Updated 9/23/2019 12:30 AM
Introduction
Materials
- 8/4/19 starter stock
- 10mL serilogical pipette
- Any red micropipette
- 24 x 20µL pipet tip
- 725mL of yamanaka media
- 25 x 50mL culture tube
- Cryostock
Procedure
Prepwork
- Place the 25 50mL culture tubes into the flow hood.
- Remove yamanaka +glucose media from the fridge and pipet 30mL of media into each culture tube using the serilogical pipette.
step 2 (-80 fridge)
- Locate where the iGEM box is in the fridge before opening (It's in the bottom left drawer and it's the first box in the drawer), then unlock the fridge and pull it out.
IMMEDIATELY CLOSE THE FRIDGE SO THE TEMP WONT INCREASE
- Pull out the cryostock (GM35)
IMMEDIATELY PUT THE IGEM BOX BACK INTO THE FRIDGE IN THE SAME SPOT AND LOCK THE FRIDGE
- Place GM35 under the flow hood.
- Grab 20µL pipet tip and gently scrape off a small amount from the cryostock.
- Eject the entire pipet tip into culture tube.
- Repeat this for 24 of the tubes.
OPEN THE FRIDGE, PULL OUT THE IGEM BOX, CLOSE THE FRIDGE, PLACE THE GM35 CRYOSTOCK BACK INTO THE IGEM BOX, OPEN THE FRIDGE AGAIN, PUT THE BOX BACK INTO THE LEFT BOTTOM SLOT, AND CLOSE THE FRIDGE. DO THIS AS QUICKLY AS POSSIBLE BECAUSE IF THE FRIDGE IS OPEN FOR MORE THAN A FEW SECONDS THE TEMP WILL RISE 20 DEGREES.
- Make sure all of the caps on the 24 culture tubes are snug but not fully closed.
- Put the entire rack of 24 tubes into Earl Gray.
Step 2 pt.2 create a negative control
- In flow hood pipet 30mL of yamanaka into the 25th culture tube.
- eject a 20µL pipet tip into the media without any k.rhaeticus
- Put this tube into Earl Gray as well.
In order to make a new stock of K. Rhaeticus we need to use the cryostock from the -80 fridge. (Due to contamination of the previous stock). We are making a 720mL of stock and separating it into 24 culture tubes to separate any future contamination. We are also making a negative control, to make be able to see if all of our stock is contaminated.