Gel Electrophoresis Lab Report 634j

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Gel Electrophoresis Drew Linarelli Honors Biology 17 May 2016 Period 9

Introduction

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A restriction enzyme is an enzyme that cuts DNA at or near a specific point known as recognition sites. They mainly work by shape to shape matching. When the enzyme meets an area of the DNA that has the same sequence of the enzyme, the enzyme causes a break in both strands of the DNA molecule. Scientists mainly use them to cut DNA into smaller fragments which could help a scientist in many ways. For instance in Genetic engineering, restriction enzymes are used to cut the DNA into fragments. They are also used to create recombinant DNA, which is when scientists bring together genetic material, such as restriction enzymes, from multiple sources creating sequences that would not otherwise be found in a genome. Restriction enzymes are also used in RFLP analysis, which is when a DNA sample is broken into fragments and then separated according to their lengths by gel electrophoresis. Through this scientists are now able to find similarities in DNA, which is very helpful for solving criminal and paternity cases.

Gel electrophoresis stated by google, “Is a laboratory method used to separate mixtures of DNA, RNA, or proteins according to molecular size. In gel electrophoresis, the molecules to be separated are pushed by an electrical field through a gel that contains small pores.” Gel electrophoresis is used to separate macromolecules like DNA and RNA, according to their size. It is also used to separate proteins according to their size and their charge. Furthermore RFLP gel electrophoresis is when electrophoresis gel separates the restriction fragments according to their size in this analysis. This method can be used for criminal and paternity cases to differentiate and identify the similarities based on the lengths concluded by the gel of the DNA strand to identify possibly the same strand of DNA or even the exact opposite strand, which could solve these types of cases.

First the purpose of this lab is we want to see if different restriction enzymes cut DNA into different sized fragments or the same sized fragments. Second we want to get practice with using tools and materials such as restriction enzymes and gel electrophoresis. Finally we want to create a logarithmic graph with known data to figure out the other lengths of our DNA fragments that were created by different restriction enzymes cutting them.

The dependent variable is the size of the fragments cut by the restriction enzymes while the independent variable is the different restriction enzymes. Then the control group is the DNA that does not consist of any restriction enzymes. The hypothesis is that the restriction enzymes will cut the DNA in different places, the smaller fragments will move faster through the gel, and the control group will not move very much if at all.

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Materials List                    

Agarose Gel TBE Buffer Solution Lambda DNA Restriction Enzymes (EcoRI, BamHI, HindIII) Micropipettes Micropipettes tips Hot plate Eddindorf reaction tubes 50 mL beakers 1000 mL flask Electrophoresis cylinder Microcentrifuge Vortex Ethidium bromide stain Loading dye Gloves Goggles Staining trays Ultraviolet light source Sharpie

Procedures Procedure A: Set up Restriction Digest 1. Label four 1.5-mL tubes, in which you will perform restrictions: B for BamHI, E for EcoRI, H for HindIII, and – for no enzyme 2. Use table below as a checklist while adding reagents to each reaction. Read down each column, adding the same reagent to all the appropriate tubes; use a fresh tip for each reagent. All groups share the same BamHi, EcoRI, HindIII enzymes at a central station 3. Pool and mix ingredients by tapping the tube bottom on the lab bench, or with a short pulse in a microcentrifuge

4 4. Incubate all reaction tubes for a minimum of 20 minutes at 37 degrees Celsius. Your teacher may instruct you to incubate the reactions for a longer period. Procedure B: Load Gel 1. Add 1 mean of loading dye to each reaction tube. Mix dye with digested DNA by tapping tube on lab bench, or with a pulse in microcentrifuge 2. Use micropipette to load contents of each reaction tube into a separated well in gel, aligned as illustrated in ideal Restriction Digest of Lambda DNA. a)

Steady pipet over well using two hands

b) Be careful to expel any air in micropipette tip end before loading gel. (if air bubbles form “cap” over well, DNA loading dye will flow into buffer around edges of well.) c) Dip pipet tip through surface of buffer, position it over the well, and slowly expel the mixture. Sucrose in the loading dye weighs down the sample, causing it to sink to the bottom of the well. Be careful not to punch tip of pipet through bottom of gel. Procedure C: Electrophorese 1. Close top of electrophoresis chamber and connect electrical leads to an approved power supply, anode to anode (red-red) and cathode to cathode (blackblack). Make sure both electrodes are connected to same channel of power supply. 2. Turn power supply on and set voltage as directed by your instructor. Shortly after current is applied, loading dye can be seen moving through gel toward positive pole of electrophorese apparatus. 3. The loading dye will eventually resolve into two bands of color. The fastermoving, purplish band is the dye bromophenol blue; the slower moving, aqua band is xylene cyanol. Bromophenol blue migrates through gel at same rate as a DNA fragment approximately 300 base pairs long. Xylene cyanol migrates at a rate equivalent to approximately 2000 base pairs. 4. Allow DNA to electrophorese until the bromophenol blue band nears the end of the gel. Your instructor may monitor the progress of electrophoresis in your absence; in that case, omit steps 5 and 6. 5. Turn off power supply, disconnect leads from the inputs, and remove top of electrophoresis chamber. 6. Carefully remove casting tray and slide gel into staining tray labeled with your group name. take your get to your instructor for staining.

Results Gel Electrophoresis lab

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This is the gel on a light box that I examined to find all of my measurements and results for this lab.

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Ideal Restriction Digest of Lambda DNA (HindIII) 1.6

1.4 f(x) = - 0.01x + 1.9 1.2

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Fragments size (log kbp)

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Distance travelled by fragmets (mm)

This graph shows the results of the HindIII data generating a best fit line. I also used the equation at the top to find the base power of my other two variables.

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This chart shows the distance and the actual base pairs for HindIII. The information in the middle of the chart was also used to create the graph on the previous page. The info is the log of kbp. Distance is in mm.

This chart shows the distance, the calculated base pairs, and the actual base pairs for EcoRI. Distance is in mm.

This chart shows the distance, the calculated base pairs, and the actual base pairs for BamHI. Distance is in mm.

This chart shows the distance and calculated base pairs of an area with no restriction enzymes. Which is the control group in this lab. Distance is in mm.

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Analysis The hypothesis was that the restriction enzymes will cut the DNA in different places, the smaller fragments will move faster through the gel, and the control group will not move very much if at all. Therefore my hypothesis was absolutely correct. I was shown that the restriction enzymes really do cut the DNA in different locations. I also found out that the smaller fragments move the farthest containing the least amount of base pairs, especially shown in “HindIII” of the Gel Electrophoresis picture on page five. However I had some sources of error while doing the lab. We did not actually use real gel to measure these results because the DNA fragments did not show up in my gel due to an unknown problem, so all we used was a picture, which is on page five. Therefore is it was hard to measure the exact points on the picture, which is the reason why some of my calculated base pairs are not incredibly accurate to the actual base pairs.

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References (Scienceclarified) Genetic Engineering using restriction enzymes source, http://www.scienceclarified.com/scitech/Genetics/Genetic-Engineering.html

(google) Google used in-text citation for how Gel electrophoresis works, https://www.google.com/?gws_rd=ssl#q=gel+electrophoresis

(biology-pages) Information on restriction enzymes source, http://www.biology-pages.info/R/RestrictionEnzymes.html

(biotechlearn) Gel electrophoresis information source, http://biotechlearn.org.nz/themes/dna_lab/gel_electrophoresis

(kau.edu) Mutations done by restriction enzymes source, http://www.kau.edu.sa/Files/0002923/Files/18591_Restriction%20Fragment %20Length%20Polymorphism.pdfa

Lab manual procedures source, Carolina Biological supply company, 2700 York Road, Burlington, North Carolina 27215, “DNA Restriction Analysis”