MOHAWK COLLEGE OF APPLIED ARTS AND TECHNOLOGY CHEMICAL AND ENVIRONMENTAL TECHNOLOGY DEPARTMENT. Lab Report ROOM NO: FE E309

Transcription

1 MOHAWK COLLEGE OF APPLIED ARTS AND TECHNOLOGY CHEMICAL AND ENVIRONMENTAL TECHNOLOGY DEPARTMENT Lab Report ROOM NO: FE E309 EXPERIMENT NO : 4 TITLE : UV Spectrophotometric Analysis of DNA Submitted by Class Partners Instructor : Lyndsay Grover : BIOL lab. : Awatif Hagelamin : Farag Soliman Date lab performed : March 10 th, 2011 Date of submission : March 17 th, 2011 FENNELL CAMPUS HAMILTON, ONTARIO 1/6

2 Purpose: The purpose of this experiment was to use ultraviolet spectrophotometry to analyze DNA to determine the quantity and purity of DNA in the sample. By observing the different absorbance levels associated with DNA and RNA we can determine the purity of the sample when calculating absorbance ratios. If there is discrepancy in the ratios we are able to determine whether or not there is protein contamination present in the sample. Apparatus: - Test tubes - Micropipette - Cuvettes - CECIL CE 2021 spectrophotometer - Reagents: DNA from experiment 3, TBE buffer Safety Guidelines: No harmful reagents are used in this experiment. Procedure: 1. Remove onion skin DNA sample from the freezer. Allow to warm up for 20 minutes. Mix well with 10uL micropipette. 2. Turn on the CECIL CE 2021 spectrophotometer and the UV (deuterium) lamp 20 minutes prior to use. Familiarize yourself with the operating instructions of the spectrophotometer before proceeding. 3. Set the spectrophotometer to read absorbance and set the wavelength to 260nm. 4. Dilute an 80 µl aliquot of your DNA stock solution with the sterile TBE buffer provided. The final volume of your dilution should be 2400µL for a 1/30 dilution which is usually a reasonable starting dilution. Use the appropriate micropippeters. 5. Remove the matched quartz cuvettes from their box. Handle them only by the frosted sides and do not touch the optical surfaces. Rinse each carefully with deionized water, then carefully shake the water droplets out. Wipe the outside of the optical surfaces of each cuvette with lens paper to remove dirt and fingerprints. 6. Zero the spectrophotometer before you read the absorbance of your DNA. Place the 1mL of TBE buffer in the quartz cuvette and wipe the optical surfaces with a Kimwipe. Make sure that you orient the cuvette with the optical surface in the path of the light or it will not transmit light. 7. Adjust the spectrophotometer to 0 absorbance (100%T) 8. Remove the background (blank) cuvette and place it in the cuvette rack. 9. Transfer your diluted DNA sample into the second cuvette and wipe the optical surfaces with a Kimwipe. Place the cuvette in the chamber and close the door. The display will show the absorbance of your sample. An absorbance between 0.1 and 1.0 is generally with the accurate reading range of most spectrophotometers. If the value is outside of the valid range, re-dilute your sample so that it falls within this range. Record your reading at 260nm and the dilution of the sample in table 1. Remove the cuvette containing the sample and place it in the cuvette rack. 10. To determine the A 280 of the diluted DNA sample, change the wavelength of the spectrophotometer to 280nm. 11. Re-zero the spectrophotometer using the cuvette containing the TBE buffer. 2/6

3 12. Place the cuvette containing the sample in the spectrophotometer, close the door and record the A 280 of the diluted DNA sample in your data table. 13. Determine the A 234 and the A 320 of the diluted DNA sample following the same procedure outlined in step Re-zero each time with the blank. 14. Store the baseline for the reference (blank) using the scanning procedure. Refer to Cecil manual. Store baseline from 220 to 320 nm with a speed of 600nm/min. 15. Scan your DNA sample from 220 to 320nm, taking absorbance readings every 10nm (scale) from 220 to 320nm to clarify the absorbance peaks and minimums. Set the absorbance min at 0 and the max at 2. Set the speed at 600nm/min.. Calculations: Corrected A 260 = /6 = Final Concentration= (0.714)(50µg/mL) = 35.7µg/mL A 260 :A 280 = 0.119/0.050 = 2.38 A 234 :A 260 = 0.090/0.119 = Data: Table 1: DNA Sample Dil. A 260 A 280 A 234 A 320 A 260 /A 280 A 234 /A 260 Final Concentration (µg/ml) 260uL 1/ /6

4 Observations: -Spectrophotometer: UV-VIS - 1/30 dilution not in range, changed to 1/31 dilution by adding 160µL. 2320µL > 2480µL -1/31 dilution not in range, changed to 1/32 dilution by adding 80µL. -1/32 dilution not in range, added 160µL to make it 1/34 dilution -1/34 dilution not in range, added 320 µl, 1/38 - realized was diluting incorrectly. Should have been making it less dilute rather than more - final dilution, 260µL DNA 1520µL TBE, 1/6 dilution Discussion: This lab is a good introduction for biotechnology students to DNA analysis and understanding the different methods available to determine the quantity and quality of DNA present in their sample. In Lab 2 we used Gel electrophoresis to determine the length of DNA strands found in our sample, when paired with this lab we can determine just how much DNA is present, and the size of the sample we are working with. This lab was a rather difficult one to perform mostly due to the fact that it required a great amount of trial and error when trying to find the right concentration of DNA. In our experiment we ended up going the wrong the direction and continuously diluting it more when we should have been making the concentration smaller. Eventually we realized the error in our ways and through more trial and error was able to find the correct dilution that brought us in range. This part of the experiment would come more easily in the future with practice and better understanding with how to read the transmission readings in terms of the DNA quantity. Sources of Error: Possible Sources of Human Error: Improper mixing of DNA. Improper dilution of DNA. Loss of sample during transfer. Contamination during transfer. Possible Sources of Equipment Error: Spectrophotometer improperly calibrated. Dirty cuvettes. Possible Sources of Experiment Error: Improper TBE buffer concentration. Post Laboratory Questions: 1. The absorbance at 320nm is If ignoring the negative it is not lower than 5% of the 260nm reading and there may be particulates within the solution. Though the negative may make this reading invalid. 2. Corrected A 260 = Concentration = 35.7µg/mL 3. A 260 :A 280 = A 230 :A 260 = When reviewing the A 260 :A 280 ratio it is above the range of a pure DNA sample, since it is above the 1.9 range that means that it is contaminated with RNA. When reviewing the A 234 :A 260 ratio it is greater than 0.50 so that means it is contaminated with protein. So this is not a very pure sample due to the fact that is it contaminated with both RNA and protein. 6. The fact that impurities are present means that this may have occurred in a few steps. The RNA contamination could have taken place during the DNA extraction. The protein contamination could have taken place during the extraction as well as the transfer if it came in contact with any other substances or human contact. 4/6

5 Conclusion: When taking into account the ratios and absorbance readings it is clear that this sample contains impurities and is not a pure sample of DNA. Looking at the ratios for A 260 :A 280 and A 234 :A 260 it clearly shows that the sample contains both RNA and protein contaminates. Also when taking in the final concentration into consideration this sample did not contain a large amount of DNA. Although our results were not successful we still were able to gain an understanding of the process used to analyze DNA via ultraviolet spectrophotometry and how the absorbance readings and their designated ratios relate to purity. We were able to clearly observe the quantity and quality of the DNA sample be determined by the readings and calculations. 5/6

6 References: Biotechnology, An Introduction S.R. Barnum, 2 nd Edition, Biotechnology 1, Laboratory Manual. Mohawk College, Custom Courseware, /6