Introduction:
All living organisms contain DNA. DNA extraction is a basic procedure of isolating the DNA from other cellular substances. It is also useful for the detection of bacteria and viruses from the environment. A pure sample of DNA that extracted from human cells can be used to test for a genetic disease, study a gene involved in cancer, body identifications or analyze forensics. Chelex resin beads used to remove the ions out of the DNA sample to prevent ions interfered with the PCR reaction. The DNA in cheek cells is identical to the DNA in blood cells.
All living organisms contain DNA. DNA extraction is a basic procedure of isolating the DNA from other cellular substances. It is also useful for the detection of bacteria and viruses from the environment. A pure sample of DNA that extracted from human cells can be used to test for a genetic disease, study a gene involved in cancer, body identifications or analyze forensics. Chelex resin beads used to remove the ions out of the DNA sample to prevent ions interfered with the PCR reaction. The DNA in cheek cells is identical to the DNA in blood cells.
Materials:
Part A: 0.9 % Sodium Chloride (NaCl), a 1.5ml microfuge tube #2 containing 200μl of 5% Chelex resin beads, paper cup, 50ml centrifuge tube capped with rack, 1.5ml microfuge tubes with rack, micropipettes and tips, serologital pipettes and electric pipettor, balanced centrifuge, vortex mixer, water bath incubator, ice bath.
Run gel: 0.8% agarose, flask, paper towel, graduated cylinder, loading dye, Ethidium Bromide (EtBr), micropipette and tips, gel tray, gel box, parafilm, microwave, digital analytical balance, TAE buffer, paper towel, Cheek-cell DNA sample and UV transilluminator.
Part B: 1.5ml Microfuge tube, 15ml centrifuge tube capped and racks, micropipettes and tips, 4M Sodium Chloride (NaCl), 10% SDS, cold 70% and 95% Ethanol (EtOH), TE buffer, paper towel and cotton swab, TAE buffer.
Part A: 0.9 % Sodium Chloride (NaCl), a 1.5ml microfuge tube #2 containing 200μl of 5% Chelex resin beads, paper cup, 50ml centrifuge tube capped with rack, 1.5ml microfuge tubes with rack, micropipettes and tips, serologital pipettes and electric pipettor, balanced centrifuge, vortex mixer, water bath incubator, ice bath.
Run gel: 0.8% agarose, flask, paper towel, graduated cylinder, loading dye, Ethidium Bromide (EtBr), micropipette and tips, gel tray, gel box, parafilm, microwave, digital analytical balance, TAE buffer, paper towel, Cheek-cell DNA sample and UV transilluminator.
Part B: 1.5ml Microfuge tube, 15ml centrifuge tube capped and racks, micropipettes and tips, 4M Sodium Chloride (NaCl), 10% SDS, cold 70% and 95% Ethanol (EtOH), TE buffer, paper towel and cotton swab, TAE buffer.
Methods:
Part A: To extract DNA from human cells. Labeled 50ml centrifuge tube capped with 50ml of 0.9% NaCl (Saline), labeled two 1.5ml microfuge tubes with name and tube #1 and tube #3. Pipetted 50ml of 0.9% NaCl (Saline) to a 50ml centrifuge tube, then transferred 10ml of saline to a new paper cup, swish the saline inside the mouth for 30 seconds to rinse off cells lining to the saline solution, spit the saline from the mouth to a paper cup and transferred 1ml of the cell-saline liquid into microfuge tube #1 labeled and tighten tube cap, then placed tube #1 to a balanced centrifuge for high speed 1 minute. Poured the supernatant from tube #1 out and added 30μl of 0.9% saline solution to resuspend the cells pellet in tube #1, made sure all the cells pellet mixed with saline solution, then pipetted 30μl of the cells suspension from tube #1 to tube #2 that containing 200μl of 5% Chelex beads and put tube #2 to vortex to mix well. Placed tube #2 with a locking cap and float boat in water bath incubator at 99°C for 10 minutes, and then quickly transferred to ice bath for 1 minute. Put tube #2 to vortex, then placed in the balanced centrifuge for high speed 1 minute. Used micropipette with a new tip to extract 60μl of Cheek-cell DNA sample without Chelex beads to the new tube #3 labeled. Stored the Cheek-cell DNA sample tube #3 in refrigerator at 4°C temperature (long-term storage at -20°C temperature). Run gel electrophoresis to detect the presence of DNA using the same steps in experiment #10 methods section.
Part B: Labeled a 15ml centrifuge tube (tube #1) and a 1.5ml microcfuge tube (tube #2) with date and initial. Pipetted 0.5ml of 4M NaCl to tube #1, used a cotton swab into the mouth to collect cells sample, then submerged the cotton swab in tube #1 that containing 0.5ml of 4M NaCl, added 0.5ml of 10% SDS and mixed gently (did not shake). Used micropipette with a new tip to transfer 0.5ml of cold 95% EtOH to a microfuge tube #2 labeled and 1ml of cold 95% EtOH to centrifuge tube #1. Made sure the cloudy white was off from cotton swab in tube #1, then discarded a cotton swab and slowly pipetted out 0.7ml of cloudy white liquid from tube #1 to tube #2 that containing 0.5ml of cold 95% EtOH. Closed tube #2 cap tightly and put in balanced centrifuge for 5 minutes. Discarded the supernatant from tube #2 and used new pipette tip to add 0.7ml of cold 70% EtOH to tube #2 and put it in the balanced centrifuge for 5 minutes, then discarded the supernatant and used new pipette tip to add 0.7ml of cold 70% EtOH to tube #2 and discarded ethanol. Dabbed tube #2 lip to paper towel and allowed to dry the DNA completely, then resuspended the pellet with 50ml TE buffer (amount depends on the size of the pellet) to tube #2, made sure DNA off the wall of tube and dissolved with TE buffer and stored in refrigerator at 4°C. Run gel electrophoresis to detect the presence of DNA using the same steps in experiment #10 methods section.
Results/Calculations: Part A: To extract DNA from human cells. Labeled 50ml centrifuge tube capped with 50ml of 0.9% NaCl (Saline), labeled two 1.5ml microfuge tubes with name and tube #1 and tube #3. Pipetted 50ml of 0.9% NaCl (Saline) to a 50ml centrifuge tube, then transferred 10ml of saline to a new paper cup, swish the saline inside the mouth for 30 seconds to rinse off cells lining to the saline solution, spit the saline from the mouth to a paper cup and transferred 1ml of the cell-saline liquid into microfuge tube #1 labeled and tighten tube cap, then placed tube #1 to a balanced centrifuge for high speed 1 minute. Poured the supernatant from tube #1 out and added 30μl of 0.9% saline solution to resuspend the cells pellet in tube #1, made sure all the cells pellet mixed with saline solution, then pipetted 30μl of the cells suspension from tube #1 to tube #2 that containing 200μl of 5% Chelex beads and put tube #2 to vortex to mix well. Placed tube #2 with a locking cap and float boat in water bath incubator at 99°C for 10 minutes, and then quickly transferred to ice bath for 1 minute. Put tube #2 to vortex, then placed in the balanced centrifuge for high speed 1 minute. Used micropipette with a new tip to extract 60μl of Cheek-cell DNA sample without Chelex beads to the new tube #3 labeled. Stored the Cheek-cell DNA sample tube #3 in refrigerator at 4°C temperature (long-term storage at -20°C temperature). Run gel electrophoresis to detect the presence of DNA using the same steps in experiment #10 methods section.
Part B: Labeled a 15ml centrifuge tube (tube #1) and a 1.5ml microcfuge tube (tube #2) with date and initial. Pipetted 0.5ml of 4M NaCl to tube #1, used a cotton swab into the mouth to collect cells sample, then submerged the cotton swab in tube #1 that containing 0.5ml of 4M NaCl, added 0.5ml of 10% SDS and mixed gently (did not shake). Used micropipette with a new tip to transfer 0.5ml of cold 95% EtOH to a microfuge tube #2 labeled and 1ml of cold 95% EtOH to centrifuge tube #1. Made sure the cloudy white was off from cotton swab in tube #1, then discarded a cotton swab and slowly pipetted out 0.7ml of cloudy white liquid from tube #1 to tube #2 that containing 0.5ml of cold 95% EtOH. Closed tube #2 cap tightly and put in balanced centrifuge for 5 minutes. Discarded the supernatant from tube #2 and used new pipette tip to add 0.7ml of cold 70% EtOH to tube #2 and put it in the balanced centrifuge for 5 minutes, then discarded the supernatant and used new pipette tip to add 0.7ml of cold 70% EtOH to tube #2 and discarded ethanol. Dabbed tube #2 lip to paper towel and allowed to dry the DNA completely, then resuspended the pellet with 50ml TE buffer (amount depends on the size of the pellet) to tube #2, made sure DNA off the wall of tube and dissolved with TE buffer and stored in refrigerator at 4°C. Run gel electrophoresis to detect the presence of DNA using the same steps in experiment #10 methods section.
Part A: To prepare 50ml of 0.9% NaCl solution used a formula % Mass/volume:
0.9g x 50ml = 0.45g of NaCl
100ml
To prepare to make 60ml of 0.8 % agarose:
0.8g x 60ml = 0.48g of agarose
100ml
Part B: To prepare to make 50ml of 0.8% agarose:
0.8g x 50ml = 0.4g of agarose
100ml
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