Alu PCR Lab
1). Purpose - Objectives
- List and explain the importance of each component of PCR
- Compare PCR to cellular DNA replication
- Associate the temperature changes with the cycling steps of PCR
2). Materials
- 9% saline solution - IXTAE
- micropipettes/tips - Gel chambers/molds
- waste container - Load dye
- microcentrifuge - Chelex
- microcentrifuge tubes - Racks
- PCR tubes - H2O
- Agarose - + Control DNA
3). Procedure
1). DNA Preparation Using a Saline Mouthwash
- Gargle 10mL of saline solution for 30 seconds
- Spit saline into a cup and swirl it to mix up the cells
- Label a 1.mL microfuge tube with some type of identification
- Pipet 1-1.5 mL of the saline suspension into the labeled microfuge tube
- Microcentrifuge the tube for 1 minute.
- Pour off the supernatant into your cup, without loosing your cell pellet
- You need about 0.1mL of saline covering it, and resuspend the cell by racking or flicking the tube
- Take 0.05mL of the cell suspension and add it to the Chelex tube
- Place your tube into a heat block for 10 minutes
- Record the location of your tube
- Remove your tube from the heat block and open it to release pressure, then close it and set it in the centrifuge for a minute
- Take a another microfuge tube and label it, include writing DNA on the tube
- Pipet 0.05mL of supernatant from Chelex/DNA tube into the other labeled tube (using a P-200 pipette and be careful to not remove any Chelex beads)
- Place tube in the class rack
2). Polymerase Chain Reaction
- Label a new tiny PCR tube
- Pipet 0.02mL of Master Mix into the tube
- Add 0.02mL of Primer Mix into the same tube, but with a new pipet tip. Mix around
- Add 0.01mL of your extracted DNA into the the same PCR tube, with another new tip
* two classmates set up a positive control reaction
* two classmates set up a negative control reaction
- your PCR tube should have about 0.05mL of substance in it.
3). Gel Electrophoresis Procedure
- 50mL of 1XTAE + 1g agarose
- heat until dissolved
- pore in the mold when it appears to be cool
- List and explain the importance of each component of PCR
- Compare PCR to cellular DNA replication
- Associate the temperature changes with the cycling steps of PCR
2). Materials
- 9% saline solution - IXTAE
- micropipettes/tips - Gel chambers/molds
- waste container - Load dye
- microcentrifuge - Chelex
- microcentrifuge tubes - Racks
- PCR tubes - H2O
- Agarose - + Control DNA
3). Procedure
1). DNA Preparation Using a Saline Mouthwash
- Gargle 10mL of saline solution for 30 seconds
- Spit saline into a cup and swirl it to mix up the cells
- Label a 1.mL microfuge tube with some type of identification
- Pipet 1-1.5 mL of the saline suspension into the labeled microfuge tube
- Microcentrifuge the tube for 1 minute.
- Pour off the supernatant into your cup, without loosing your cell pellet
- You need about 0.1mL of saline covering it, and resuspend the cell by racking or flicking the tube
- Take 0.05mL of the cell suspension and add it to the Chelex tube
- Place your tube into a heat block for 10 minutes
- Record the location of your tube
- Remove your tube from the heat block and open it to release pressure, then close it and set it in the centrifuge for a minute
- Take a another microfuge tube and label it, include writing DNA on the tube
- Pipet 0.05mL of supernatant from Chelex/DNA tube into the other labeled tube (using a P-200 pipette and be careful to not remove any Chelex beads)
- Place tube in the class rack
2). Polymerase Chain Reaction
- Label a new tiny PCR tube
- Pipet 0.02mL of Master Mix into the tube
- Add 0.02mL of Primer Mix into the same tube, but with a new pipet tip. Mix around
- Add 0.01mL of your extracted DNA into the the same PCR tube, with another new tip
* two classmates set up a positive control reaction
* two classmates set up a negative control reaction
- your PCR tube should have about 0.05mL of substance in it.
3). Gel Electrophoresis Procedure
- 50mL of 1XTAE + 1g agarose
- heat until dissolved
- pore in the mold when it appears to be cool