You will use the extracted genomic dna as the target

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Unformatted text preview: suspend your isolated cheek cells in the InstaGene matrix and incubate them at 56°C for 10 minutes. This preincubation step helps to soften plasma membranes and release clumps of cells from each other. The heat also inactivates enzymes, such as DNases, which can degrade the DNA template. After this 10 minute incubation period, place the cells in a boiling (100°C) water bath for 5 minutes. Boiling ruptures the cells and releases DNA from their nuclei. You will use the extracted genomic DNA as the target template for PCR amplification. *The introductory information and protocol has been adapted from “Biotechnology Explorer™Chromosome 16: PV92 PCR Informatics Kit Manual which is a product of Bio-Rad. Duplication of any part of this document is permitted for classroom use only. 1 Bio 05LA – Fall Quarter 2012 Lab 8 Procedure Lab Period 1 - Cheek Cell DNA Template Preparation 1. Obtain a cup containing saline solution from your instructor and go to your lab bench. Pour the saline into your mouth and rinse vigorously for 30 seconds. Expel the saline back into the cup. 2. Labe l the 1 .5 m l m icro tes t tube (a) o n the b ench with your seat number. Label one of the screwcap tube (b) containing 200 µl of InstaGene matrix in the ice bucket with your seat number. Return the screwcap tube to the ice bucket after labeling. (a) (b) 3. Transfer 1 ml of your saline rinse into the micro test tube (a) using a 1 ml pipet. Make sure you are not adding your saline rinse to the screwcap tube (b) in the ice bucket. 4. Along with the rest of your classmates, spin your tube in a balanced centrifuge at full speed for 2 minutes. When the centrifuge has completely stopped, remove your tube. You should see a match-head sized pellet of whitish cells at the bottom of the tube. If you don’t see a pellet of this size, decant the saline, refill your tube with more of your oral rinse, and repeat the spin. 5. After pelleting your cells, pour off the saline. Being careful not to lose your pellet, blot your tube briefly on a paper towel or tissue. It’s OK for a small amount of saline (< 50 µl, about the s am e s ize as you r p ellet) to remai n i n the bottom of the tube. 6. Resuspend the pellet by vortexing or flicking the tube so that no clumps of cells remain. 7. Using a sterile Pasteur pipet, transfer all of your resuspended c ell s to th e screwca p tub e containing InstaGene. 8. Screw the cap tightly on the tube. Shake or vortex to mix the tube contents. 2 (a) Centrifuge Bio 05LA – Fall Quarter 2012 Lab 8 9. W he n a l l m embers o f your team h ave collected their samples, place the tubes in the micro test-tube holder and incubate at 56°C for 10 minutes in a water bath. At the halfway point (5 minutes), shake or vortex the tubes gently, then place back in the 56°C water bath for the remaining 5 minutes. Water bath 56°C, 10 min Very Hot! 10. Remove the tubes, shake or vortex, and place the tubes i n a b oilin g water bath (100°C). Incubate at 100°C for 5 minutes. Be careful, the steam is very hot! Water bath 100°C, 5 min 11. Remove the tubes from the boiling water bath and shake or vortex the contents to resuspend. Pellet the matrix by spinning at full speed for 5 minutes in a centrifuge. Centrifuge 12. Place your screwcap tube in the TA’s ice bucket. The TA will mix which contains the nucleotides, primers, magnesium ions. Buffer and Taq Polymerase. The tubes are then placed in the thermal cycler for 40 cycles of amplification. Once the program has run the PCR reactions will be frozen and stored until the next lab meeting. 3 Biology 05LA – Fall Quarter 2012 Lab 4 – page 1 LAB 4 -- HYPOTHESIS-BASED SCIENCE; AN INTRODUCTION TO THE HYPOTHETICO-DEDUCTIVE APPROACH TO PROBLEM SOLVING. Science has been described as a “way of knowing”, that is, it is an activity whereby we learn about our world. This learning can be achieved by two different approaches. The first applies to inquiries in new or poorly understood areas and is referred to as discovery or descriptive science. Here, data is collected in the form of careful observations of a phenomenon of interest. This data is then carefully analyzed and recorded in a retrievable manner. As more and more data is collected, generalizations about this phenomenon can be made and our understanding thus increases. The type of reasoning by which this understanding was achieved is referred to as inductive because larger generalizations are derived from many smaller specific observations. In contrast, hypothesis-based science deals with questions in areas that are better understood. Here, problem solving begins with a well-defined question. The next step involves consideration of what is known about related phenomena and how we came to know these things. This information is then used as the basis for formulating a hypothesis, or possible answer to the question. The order in which this factual information is assembled is determined with the use of deductive reasoning. That is, it is ordered in a manner that places more general knowledge fir...
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This note was uploaded on 08/27/2013 for the course BIO BIOL05LA taught by Professor Abbottl during the Fall '12 term at UC Riverside.

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