Exercise 4 Analysis

Exercise 4 Analysis - 1. In essence the agarose gel is like...

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1. In essence the agarose gel is like a maze for the strands of DNA to travel through. The longer the strand of DNA that is traveling through the gel the harder it is for it to travel through the matrix like structure of the gel. In this case the DNA is negatively charged because of its negatively charged phosphate groups on the back bone of the molecule, so DNA will move toward the positively charged anode. What will determine the distance traveled by the DNA strand is the size the molecule, the shorter the strand the farther it will go the longer the strand the shorter the distance it will move. The 6X loading dye solution insures that the strands of DNA remain in the wells. 2. The agarose gel has pores of one or more defined sizes, so large molecules move more slowly and less far in the gel than do smaller molecules. To make the gel, first it must be made into a solution by melting it to a solution using a microwave, then pouring the solution into the casting tray and letting it sit with a comb in place so that it can once again solidify. This gel is suitable for separating larger molecules such as DNA and most RNAs. A 0.7% gel will show good separation (resolution) of large DNA fragments (5– 10kb) and a 2% gel will show good resolution for small fragments (0.2–1kb). 3. The TAE buffer has a lower buffering capacity compared to most other buffers which enables the DNA to run faster through the gel, the higher the concentration the faster the bigger sections of DNA can run through the gel, because the higher the concentration the higher the charge differential. 4. By adding Ethidium Bromide to the DNA molecules it is possible to stain them so that when looked at under a UV light the bands of the DNA you light up enabling for easy identification. 5. The human DNA buccal samples contained either the TE buffer, the Chelex beads, primer mix, and the master mix (which contained Taq Polymerase, MgCL2, DNTPs, and 2X buffer) The final volume of the reaction mix in the tube was 20 µL and the final concentration of the 2X Taq Buffer is 0.8M since our final volume was 20 µL and the total final volume was 25 µL, so (20 µL / 25 µL) = 0.8M 6. There are several components that are necessary to make a PCR experiment a success. You need DNA strand template, which is the section of DNA that is desired to be amplified. Two primers are necessary to adhere the to the DNA template strand followed by the Taq Polymerase that enables for the addition of the dNTPs that must also be present in the procedure. Finally you must have the appropriate buffers and cofactors that will help facilitate the ease at which the procedure is done. There are three main steps which are the denaturing step where the temperature must be raised to 94ºC so that the Hydrogen bonds can be broken. The annealing step is where the temperature is lower to 54ºC, just enough for the primers to attach 1
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to the denatured DNA strands. The final main step is the extension step
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This note was uploaded on 05/06/2010 for the course BIO 206L taught by Professor Unknown during the Spring '08 term at University of Texas.

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Exercise 4 Analysis - 1. In essence the agarose gel is like...

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