Part B DNA Isolation 1 The tube containing the swab LS solution and the PK

Part b dna isolation 1 the tube containing the swab

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Part B – DNA Isolation 1. The tube containing the swab, LS solution and the PK solution was placed in a 60 0 C water bath for 1 hour then vortex briefly. 2. The liquid in the tube was then transferred into a 1.5ml centrifuge tube using a sterile pipette tip. 3. 400µl of the CT solution was the added to the tube and vortex briefly.
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4. The tube was placed in a microcentrifuge and was put to spin at 4000rpm for 15 minutes to pellet the DNA. 5. All the supernatant was removed carefully with a pipette tip making sure not to disturb the DNA pellet. 6. The tube was re-spinned briefly and any remaining liquid was removed. 7. 150µl of the TE solution was added to the tube. 8. The tube was left for at least 5 minutes at room temperature for the DNA to re-hydrate, and vortex briefly. 9. The DNA was stored at -20 0 C for two weeks. ADDITIONAL 1. 5µl of the DNA was diluted in 995µl of distilled water. 2. 2ml of distilled water was then added to the DNA again. 3. The purity of the DNA sample was determined by spectrophotometry reading at A260 and A280. 4. The spectrophotometer was turned on to warm up for about 15 minutes. 5. One of the cuvette was filled with distilled water. This was the blank. And was placed at position 1 of the cuvette in the spectrophotometer. 6. All the samples were filled with the diluted DNA sample and were placed in the spectrophotometer. 7. The wavelength for the absorbance was adjusted to 260nm. 8. Results were recorded and step 7 above was repeated with an absorbance of 280nm. RESULTS: Table Showing the Absorptions at 260nM and 280nM for each of the DNA samples. STUDENT ABSORPTION 260nm/ 280nm 260 nM 280 nM Blank 0.0765 0.0655 1.2 1 0.0806 0.0682 1.2 2 0.0729 0.0617 1.2
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3 0.0760 0.0647 1.2 4 0.0784 0.0664 1.2 5 0.0780 0.0672 1.2 6 -0.0461 -0.0426 1.1 CALCULATIONS: Nucleic Acids, DNA and RNA, absorb at 260nM and proteins absorb at 280Nm. A ratio 260nM/280nM of 1.8-1.9 indicates pure DNA and a ratio of 1.9-2.0 indicates pure RNA. Blank ratio= 0.0765/0.0655 = 1.2 Student 1 ratio = 0.0806/ 0.0682 = 1.2 Student 2 ratio = 0.0729/ 0.0617 = 1.2 Student 3 ratio = 0.0760/ 0.0647 = 1.2 Student 4 ratio = 0.0784/ 0.0664 = 1.2 Student 5 ratio = 0.0780/ 0.0672 = 1.2 Student 6 ratio = -0.0461/ -0.0426 = 1.1
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DISCUSSION: Only a few reagents were used to extract DNA from cells: lysis buffer, protease k, a capture buffer and TE. Lysis buffer broke open the cheek cells to expose DNA and cellular proteins. This explains why the sample in this experiment became clear upon addition of the lysis buffer. Protease k served to enzymatically break down the proteins in the sample. From theory a ratio of 1.8-1.9 indicated a pure DNA and a ratio of 1.9-2.0 indicates a pure RNA while a low ratio indicates that there are a lot of protein being absorbed at 280nM. From the results obtained from this lab it can be said that all samples had a lot of protein being absorbed at 280nM. Also, the use of ethanol instead of distilled water could have been a reason why the results were different. QUESTIONS & ANSWERS: QUESTIONS
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