Procedures Each group will be provided with two 20 µg double-stranded DNA oligomers A and B in STE buffer (0.1M NaCl/ Tris/ 10 mM EDTA, pH 7.4). The sequence of the two oligomers used in this experiment is: 5' GCATTGCGCAGGGCCGAG 3’ (GC rich) 3' AATGGTACGTATACTTTAT3’ (AT rich) In this experiment, you are going to identity oligomer samples A and B, GC or AT rich, by UV spectrophotometric method. 1. Pipet 1 ml of each oligomer into a 1.5 ml Eppendorf tube and label the two tubes A and B. 2. The absorption wavelength is 260 nm. Use STE buffer provided to set blank. 3. You will be provided with two cuvettes. Use separate cuvette for each DNA sample. 4. Transfer 1 ml of DNA sample A to cuvette and measure the UV absorbance at 260 nm (A260) at room temperature. Repeat this step for Sample B. 5. Transfer the DNA back to the original Eppendorf tube, close it and heat it to 45°C for 7 minutes. 6. Quickly transfer the sample from Eppendorf tube to cuvette, and measure the A260 as in step 3 immediately. 1 7. Repeat step 4 and 5 by heating the sample to 60°C, 75°C and 95°C, and measure their A260- Before heating the sample at 75°C and 95°C, use a small piece of parafilm to wrap around the cap of the Eppendorf tube to prevent the cap from pop open. Slightly tap the tube if sample is condensed on the cap. 8. After 95°C, cool down the tube to 75°C, and 60°C and then 45°C for 7 minutes each, and measure the A260 at each temperature. 9. Do the heating and measuring A260 of Samples A and B in parallel to save time and maintain consistency.
Procedures Each group will be provided with two 20 µg double-stranded DNA oligomers A and B in STE buffer (0.1M NaCl/ Tris/ 10 mM EDTA, pH 7.4). The sequence of the two oligomers used in this experiment is: 5' GCATTGCGCAGGGCCGAG 3’ (GC rich) 3' AATGGTACGTATACTTTAT3’ (AT rich) In this experiment, you are going to identity oligomer samples A and B, GC or AT rich, by UV spectrophotometric method. 1. Pipet 1 ml of each oligomer into a 1.5 ml Eppendorf tube and label the two tubes A and B. 2. The absorption wavelength is 260 nm. Use STE buffer provided to set blank. 3. You will be provided with two cuvettes. Use separate cuvette for each DNA sample. 4. Transfer 1 ml of DNA sample A to cuvette and measure the UV absorbance at 260 nm (A260) at room temperature. Repeat this step for Sample B. 5. Transfer the DNA back to the original Eppendorf tube, close it and heat it to 45°C for 7 minutes. 6. Quickly transfer the sample from Eppendorf tube to cuvette, and measure the A260 as in step 3 immediately. 1 7. Repeat step 4 and 5 by heating the sample to 60°C, 75°C and 95°C, and measure their A260- Before heating the sample at 75°C and 95°C, use a small piece of parafilm to wrap around the cap of the Eppendorf tube to prevent the cap from pop open. Slightly tap the tube if sample is condensed on the cap. 8. After 95°C, cool down the tube to 75°C, and 60°C and then 45°C for 7 minutes each, and measure the A260 at each temperature. 9. Do the heating and measuring A260 of Samples A and B in parallel to save time and maintain consistency.
Human Anatomy & Physiology (11th Edition)
11th Edition
ISBN:9780134580999
Author:Elaine N. Marieb, Katja N. Hoehn
Publisher:Elaine N. Marieb, Katja N. Hoehn
Chapter1: The Human Body: An Orientation
Section: Chapter Questions
Problem 1RQ: The correct sequence of levels forming the structural hierarchy is A. (a) organ, organ system,...
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Transcribed Image Text:Procedures
Each
group
will be provided with two 20 µg double-stranded DNA oligomers A and B in STE buffer
(0.1M NaCl/ Tris/ 10 mM EDTA, pH 7.4). The sequence of the two oligomers used in this
experiment is:
5' GCATTGCGCAGGGCCGAG 3’ (GC rich)
3' AATGGTACGTATACTTTAT3’ (AT rich)
In this experiment, you are going to identity oligomer samples A and B, GC or AT rich, by UV
spectrophotometric method.
1. Pipet 1 ml of each oligomer into a 1.5 ml Eppendorf tube and label the two tubes A and B.
2. The absorption wavelength is 260 nm. Use STE buffer provided to set blank.
3. You will be provided with two cuvettes. Use separate cuvette for each DNA sample.
4. Transfer 1 ml of DNA sample A to cuvette and measure the UV absorbance at 260 nm (A260) at
room temperature. Repeat this step for Sample B.
5. Transfer the DNA back to the original Eppendorf tube, close it and heat it to 45°C for 7 minutes.
6. Quickly transfer the sample from Eppendorf tube to cuvette, and measure the A260 as in step 3
immediately.
1
7. Repeat step 4 and 5 by heating the sample to 60°C, 75°C and 95°C, and measure their A260.
Before heating the sample at 75°C and 95°C, use a small piece of parafilm to wrap around the
cap of the Eppendorf tube to prevent the cap from pop open. Slightly tap the tube if sample is
condensed on the cap.
8. After 95°C, cool down the tube to 75°C, and 60°C and then 45°C for 7 minutes each, and
measure the A260 at each temperature.
9. Do the heating and measuring A260 of Samples A and B in parallel to save time and maintain
consistency.
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