Alu PCR Lab
Purpose
To successfully isolate DNA from cheek cells and prepare a PCR reaction for amplification of an Alu insert.
To successfully isolate DNA from cheek cells and prepare a PCR reaction for amplification of an Alu insert.
Materials
-0.9% saline solution -micropipetts, tips
-waste container -microcentrifuge, tubes
-PCR tubes -agarose
-1x TAE -gel chambers + molds
-load dye -chelex
-racks -primer mix
-master mix -H2O
- + control DNA
-0.9% saline solution -micropipetts, 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. Swirl 0.9% saline solution in mouth for 30 sec., spit into a cup
2. Transfer 1 mL - 1.5 mL of saline/cell suspension into a microfuge tube, spin for 1 min.
3. Pour out supernatant, resuspend cells, move 50 uL of cells to a tube w/ chelex, place in heat block for 10 min.
4. Centrifuge tube for 1 min., transfer 50 uL of supernatant into a new tube, put in rack
5. Obtain new tube, add 20 uL master mix, 20 uL primer mix , and 10 uL of your DNA, place in thermal cycler
6. Briefly spin tube in centrifuge, transfer 20 uL of DNA sample to fresh tube, add 4 uL loading dye, spin again
7. Load 20 uL of DNA sample into gel well
8.
Gel Procedure (Before step 7)
1. Mix 50 mL of 1x TAE w/ 1 g of agarose, heat until dissolved
2. Pore into mold when cooled
1. Swirl 0.9% saline solution in mouth for 30 sec., spit into a cup
2. Transfer 1 mL - 1.5 mL of saline/cell suspension into a microfuge tube, spin for 1 min.
3. Pour out supernatant, resuspend cells, move 50 uL of cells to a tube w/ chelex, place in heat block for 10 min.
4. Centrifuge tube for 1 min., transfer 50 uL of supernatant into a new tube, put in rack
5. Obtain new tube, add 20 uL master mix, 20 uL primer mix , and 10 uL of your DNA, place in thermal cycler
6. Briefly spin tube in centrifuge, transfer 20 uL of DNA sample to fresh tube, add 4 uL loading dye, spin again
7. Load 20 uL of DNA sample into gel well
8.
Gel Procedure (Before step 7)
1. Mix 50 mL of 1x TAE w/ 1 g of agarose, heat until dissolved
2. Pore into mold when cooled