Ecosystems of Homewood Part IV; Cellular Respiration in Peas (Cellular…
Ecosystems of Homewood Part IV; Cellular Respiration in Peas
PCR Amplification of the 16S Ribosomal DNA Gene
Clamp lid shut with lid lock. Repeat this step and last step for 2nd sample.
Incubate both samples in boiling water for 20 minutes.
Use a sterile wire loop to move 1 entire bacterial colony into the tube of water.
Obtain 2 PCR tubes with PCR master mix.
Take a photo of each plate before you remove each colony in the next step.
Remove 1 uL from each heated bacterial sample and add it to appropriate master mix tube.
Get 2 microcentrifuge tubes; each containing 100 uL sterile water.
Put the tubes in the thermocycler.
Choose 2 colonies to identify
Cellular Respiration in Peas
Add another ball of nonabsorbent cotton to all 3 tubes.
Put another ball of absorbent cotton in and moisten with 2 drops of 15% KOH.
Label tubes and add the right stuff.
Add stoppers with serological pipettes and syringes to all tubes.
Place a ball of nonabsorbent cotton on top of the moist one.
Lay the tubes on their side for 5 minutes.
Use a Pasteur pipette to carefully drop in 4 or 5 drops of 15% KOH. Don't let the KOH touch the walls of the tube.
Add a drop of dye to the end of each serological pipette.
Take 3 empty test tubes and add a small ball of absorbent cotton to the bottom of each.
Adjust the dye blob so the top of the dye is at the 0 mark on the pipet.
Record the dye position every 2 minutes for 14 minutes on each of the 3 tubes. Movement away from the tube is positive numbers.
Be careful when disassembling. Follow directions in packet.