Alu PCR Lab
Purpose:
1. Succesfully isolate DNA from check cells
2. Prepare a PCR reaction fro amplification of an Alu insert
Materials:
9% Saline Solution
Micropipettes
Tips
Waste Container
Microcentrifuge
Microcentrifuge tubes
PCR tubes
Agarose
1x TAE
Gel chambers + molds
Load dye
Racks
Primer mix
Master mix
Deionized Water
Control DNA
Chelex
Procedure:
1. Swirl 10ml of saline arrangement in your mouth for 30 seconds and spit it into a mug to blend the cells.
2. Exchange 1000ul to 1500ul of the saline/cell suspension in your named tube
3. Turn your saline cell suspension in a microcentrifuge for a moment.
4.Make beyond any doubt there is 100ul of saline staying in the tube, flick or rack the tube to blend, resuspending the cell
5. Name a container of chelex and include 50 ul of your cell suspension into the tube.
6. Take your tube to a warmth piece station and abandon it there for 10 minutes.
7. In the wake of warming, uproot the top lock to discharge weight, then get another clean microfuge tube and mark it, compose DNA on this tube
8. Utilize a P-200 to withdraw 50 ul of supernatant from the chelex/DNA tube to your new tube. Verify that you don't exchange any chelex globules.
9. Put your DNA tube in the class rack. Your instructor will refrigerate it until you are prepared to set up your PCR intensification.
10. Take a minor PCR tube and pipet 20 ul of Master blend into PCR tube
11. Change your pipet tip and include 20ul of Primer Mix into your PCR tube
12. Include 10ul of your removed DNA into the PCR tube
13. Place your response into the warm chamber
14. Recover your PCR tube and spot it in an adjusted design in a microcentrifuge. Turn it for 10 seconds to convey the fluid to the base of the response tube.
15. Add 5ul of stacking color to your PCR tube
16. Precisely stack 15 to 20 ul of the DNA/stacking color mixture into a well in your gel.
17. One understudy ought to load 5-10ul of 100 bp step into one of the wells of every gel.
18. At the point when the majority of the examples are stacked, connect the cathodes from the gel box to the force supply, then electrophorese your specimens at 150 volts for 25-40 minutes.
19. After electrophoreses, the gels will be prepared to stain and photo.
1. Succesfully isolate DNA from check cells
2. Prepare a PCR reaction fro amplification of an Alu insert
Materials:
9% Saline Solution
Micropipettes
Tips
Waste Container
Microcentrifuge
Microcentrifuge tubes
PCR tubes
Agarose
1x TAE
Gel chambers + molds
Load dye
Racks
Primer mix
Master mix
Deionized Water
Control DNA
Chelex
Procedure:
1. Swirl 10ml of saline arrangement in your mouth for 30 seconds and spit it into a mug to blend the cells.
2. Exchange 1000ul to 1500ul of the saline/cell suspension in your named tube
3. Turn your saline cell suspension in a microcentrifuge for a moment.
4.Make beyond any doubt there is 100ul of saline staying in the tube, flick or rack the tube to blend, resuspending the cell
5. Name a container of chelex and include 50 ul of your cell suspension into the tube.
6. Take your tube to a warmth piece station and abandon it there for 10 minutes.
7. In the wake of warming, uproot the top lock to discharge weight, then get another clean microfuge tube and mark it, compose DNA on this tube
8. Utilize a P-200 to withdraw 50 ul of supernatant from the chelex/DNA tube to your new tube. Verify that you don't exchange any chelex globules.
9. Put your DNA tube in the class rack. Your instructor will refrigerate it until you are prepared to set up your PCR intensification.
10. Take a minor PCR tube and pipet 20 ul of Master blend into PCR tube
11. Change your pipet tip and include 20ul of Primer Mix into your PCR tube
12. Include 10ul of your removed DNA into the PCR tube
13. Place your response into the warm chamber
14. Recover your PCR tube and spot it in an adjusted design in a microcentrifuge. Turn it for 10 seconds to convey the fluid to the base of the response tube.
15. Add 5ul of stacking color to your PCR tube
16. Precisely stack 15 to 20 ul of the DNA/stacking color mixture into a well in your gel.
17. One understudy ought to load 5-10ul of 100 bp step into one of the wells of every gel.
18. At the point when the majority of the examples are stacked, connect the cathodes from the gel box to the force supply, then electrophorese your specimens at 150 volts for 25-40 minutes.
19. After electrophoreses, the gels will be prepared to stain and photo.