ALU PCR Lab
Purpose: To successfully isolate DNA from cheek cells and prepare a PCR reaction for amplification of an Alu insert.
Materials:
-0.9% saline solution -micropipettes, tips
-waste container -microcentrifuge, tubes
-PCR tubes -agarose
-1x TAE -gel chambers + molds
-load dye -chelex
-racks -primer mix
-master mix -H2O
- +&- control DNA
Procedure:
1. Swish 0.9% saline solution in your mouth for 30 seconds, then spit into a cup.
2. Pipette 1mL-1.5ml into a labelled microcentrifuge tube, spin in microcentrifuge for 1 minute
3. Pour off the supernatant and resuspend the cells
4. Pipette 50 uL into a new tube with Chelex
5. Place in heat block for 10 minutes
6. Spin for 1 minute, put 50 uL of supernatant into new tube and rack
7. In a new tube, pipette 20 uL of Master and Primer mix each, and 10 uL of your DNA, then put into the thermal cycler
8. Centrifuge, then pipette 20 uL of DNA and 4uL of loading dye
9. Centrifuge, then load into gel
Gel Procedure:
1. Mix 50 mL of 1x TAE with 1 g of agarose
2. Heat until dissolved
3. Pour into mold when cool (you can touch it without burning yourself)
Materials:
-0.9% saline solution -micropipettes, tips
-waste container -microcentrifuge, tubes
-PCR tubes -agarose
-1x TAE -gel chambers + molds
-load dye -chelex
-racks -primer mix
-master mix -H2O
- +&- control DNA
Procedure:
1. Swish 0.9% saline solution in your mouth for 30 seconds, then spit into a cup.
2. Pipette 1mL-1.5ml into a labelled microcentrifuge tube, spin in microcentrifuge for 1 minute
3. Pour off the supernatant and resuspend the cells
4. Pipette 50 uL into a new tube with Chelex
5. Place in heat block for 10 minutes
6. Spin for 1 minute, put 50 uL of supernatant into new tube and rack
7. In a new tube, pipette 20 uL of Master and Primer mix each, and 10 uL of your DNA, then put into the thermal cycler
8. Centrifuge, then pipette 20 uL of DNA and 4uL of loading dye
9. Centrifuge, then load into gel
Gel Procedure:
1. Mix 50 mL of 1x TAE with 1 g of agarose
2. Heat until dissolved
3. Pour into mold when cool (you can touch it without burning yourself)