Protocol 6: Gel Electrophoresis
Contents
1. ChE 130 Spring 2015 Protocol 6 Analysis of double stranded DNA with agarose gel electrophoresis Background DNA is commonly analyzed by tracking its rate of migration through a semi solid gel The sizes of DNA fragments in an experimental sample are estimated by comparing the rate of migration of these fragments or their relative locations on the gel to that of fragments of DNA of known size commonly referred to as ladders Double stranded pieces of DNA greater than 50 100 bp in length are typically analyzed by horizontal slab agarose gel electrophoresis Agarose is a linear polymer composed of alternating residues of D and L galactose joined by a 1 3 and B 1 4 glycosidic linkages Gelation of agarose results in a three dimensional mesh of channels whose diameters range from 50 nm to greater than 200 nm The rate of migration of DNA through agarose gels is determined by a number of factors including molecular size of the DNA Molecules of double stranded DNA migrate through gel matrices at a rate that is inversely proportional to the log of the number of base pairs Larger molecules migrate more slowly because of greater frictional drag and because they worm their way through the pores of the gel less efficiently than smaller molecules agarose concentration A DNA fragment of a given size will migrate at different rates through gels containing different concentrations of agarose according to the following equation2. constant voltage at approximately 12 V cm typically between 80 100 V This setting can be modified depending on whether you want the separation to occur faster or slower however we do not run gels of this size faster than 100 V Better resolution will be obtained at slower separation speeds 10 Monitor the movement of the DNA through the gel by monitoring the location of the loading dyes in the gel as it runs The DNA will move towards the anode side of the gel box red Stop the separation when the faster moving dye purple color is approximately 50 75 across the length of the gel adjusted as appropriate for the fragments you are separating Turn off the power supply and remove the gel tray from the running apparatus Control Switch Left SD Gel Imager control Right Computer control LCD Display Scan Blue White Light Switch 11 Lift the scanner lid and place it at the center of the sample tray 12 The gel imager should be turned on and will likely be in standby mode unless it was recently used In order to image DNA gels set the light switch to Blue the White light setting can be used for imaging plates All relevant buttons and switches are labelled in the figure above you shouldn t need to change any settings 13 Flip the control switch to the right Wait for an image of your gel to appear on the LCD display ChE 130 Spring 2015 14 When an image appears press Scan Wait for the CAPTURING text on the LCD d
3. f the gel try Make sure that the apparatus is level Based on the size of the DNA you will be separating on the gel determine the appropriate percentage gel to run Full length plasmids can be effectively separated using a 0 8 gel whereas smaller fragments 100 bp require a 1 5 2 gel Medium sized fragments can be effectively separated using an intermediate agarose concentration Note that these percentages are in weight gram per volume mL units Make up the gel solution in a 250 ml Erlenmeyer flask The total volume required for one gel is 35 ml Weigh out the appropriate amount of agarose using a metal spatula and weighing dishes Carefully pour the agarose into the flask Measure out 50 ml of 1X TAE in a graduated cylinder and pour the buffer into the flask over the agarose powder Mix by swirling Note that the agarose will not go into solution Cap the flask loosely with a KimWipe and put it into the microwave Typically heat on high for 20 seconds remove and swirl briefly and then heat for another 10 seconds Swirl the mixture so that the agarose is melted and then cool briefly by allowing the solution to cool slowly on the bench top or by running the flask under tap water Once the mixture has cooled for 1 2 minutes the gel mixture should still be warm to the touch add 0 5 uL of GelGreen per 50 ml gel solution Swirl to mix and pour 35 ml of the gel solution into the ChE 130 Spring 2015 gel tray Place the appropr
4. iately sized comb into place and make sure that it is situated correctly in the gel Allow the gel to cool in the tray and solidify 5 While the gel is solidifying approximately 30 minutes prepare the DNA solutions to be run For the small combs solutions of 6 10 ul are typically run for the large combs solutions of 30 ul are run these volumes are approximate Add loading buffer to each sample such that the final concentration is 1X loading buffer 6 Select the appropriate DNA ladder either 1 kb or 100 bp DNA ladder depending on the sizes of the DNA to be separated The ladder comes pre mixed with loading buffer 7 Once the gel has solidified carefully remove the black placeholders and comb Orient the gel such that the wells are closest to the cathode side of the gel box black Pour DNA Gel Running Buffer 1X TAE into the box such that the gel is completely but just covered 8 Samples should be loaded using a L20 pipetman set to the appropriate volume Mix DNA samples prior to loading by pipetting up and down briefly When loading a gel it is easiest if there are no air pockets in the pipet tip Also you will most likely have to stabilize your loading arm either on the bench top or with your other arm You ll also have to position yourself so that you can see the wells and place the pipet tip just over the well Do not insert the tip too deeply into the well as you will likely pierce through the well 9 Once loaded run the gel at
5. ing buffer composed of sucrose and two dyes bromophenol blue and xylene cyanol FF The loading buffer serves to i increase the density of the DNA sample so that it sinks into the well ii add color to the sample to simplify the loading process and iii add dyes to the sample which will migrate toward the anode at predictable rates DNA is most easily visualized by a fluorescent nucleic acid stain that binds specifically to nucleic acid molecules The stain GelGreen Phenix will be included in the agarose slab used for electrophoresis This allows for instant visualization of DNA under blue light illumination Information in this background section was adapted from Sambrook J amp Russell DW Molecular cloning a laboratory manual 3 ed Cold Spring Harbor Laboratory Press Cold Spring Harbor NY 2001 Materials TAE 1X fresh GelGreen DNA stain 100 000X DNA double distilled autoclaved water ddH2O agarose benchtop microfuge loading buffer 6X ready to load ladder stock assorted pipetmen assorted tips 100 ml graduated cylinder KimWipes gel box tray comb and power supply 1 5 mL microfuge tube Gel Running Buffer Used TAE microwave FastGene GelPic LED BOX 125 ml Erlenmeyer flask weighing dish scale Zymo spin columns 2 ml collection tubes ADB Buffer DNA Wash Buffer Method 1 Prepare the gel tray for pouring of the gel solution by placing it into the tray apparatus and 2 placing black placeholders at the sides o
6. isplay to appear and disappear this indicates that your image has already be taken and saved to the SD card 15 Flip the control switch to the left SD If using the gel imager connected to the computer 16 Go to Windows Explorer open up Removable Disk Drive and go into folder 200Media The most recent image largest number in the folder should be the image that was just taken Save it for your laboratory notebook Please note that images taken with FastGene Gel Imager cannot be quantified but can be used for image purposes If using the wireless gel imager 16 You will need to have a smartphone with the PQI Air Card app installed Go into your phone settings and under WiFi networks connect to the PQI Air Card network Next open the PQI Air Card app and select the images icon You will be able to browse through the images stored on the SD card in the imager Select the files you wish to save and either download them to your phone or choose the share option to email them to yourself Afterwards disconnect your phone from the PQI Air Card WiFi network since only a limited number of connections can be established at any one time 17 Discard gel in wastebucket and clean sample tray dry with a kimwipe Please note that images taken with FastGene Gel Imager cannot be quantified but can be used for image purposes Note the FastGene Gel Imager User Manual is also available on the Desktop Gel Extraction 1 Keep the con
7. logiou logio uo Ky t where u electrophoretic mobility of DNA t gel concentration o free electrophoretic mobility of DNA K retardation coefficient DNA conformation Superhelical circular form I nicked circular form II and linear form IHD DNAs migrate through agarose gels at different rates Under most circumstances form I will migrate faster than form III applied voltage At low voltages the rate of migration of DNA fragments is proportional to the applied voltage Note that the effective range of separation decreases as voltage increases because the mobility of high molecular weight DNA increases differentially electrophoresis buffer The composition and ionic strength of the electrophoresis buffer affects the electrophoretic mobility of DNA In the absence of ions electrical conductivity is minimal and DNA migrates slowly agarose Different classes of agarose have different resolving powers and are used for different applications We will be using standard agarose for the separations performed in this laboratory We will also be using a Tris acetate and EDTA TAE buffer for electrophoresis TAE has a relatively low buffering capacity and will become exhausted if electrophoresis is carried out for long periods of time Double stranded DNA migrates 10 faster in TAE than in other commonly used buffers and TAE has better resolving power for larger DNA fragments We will ChE 130 Spring 2015 use a load
8. new 1 5 ml microfuge tube To elute DNA add 6 25 ul of double distilled autoclaved water ddH2O to the spin column The choice in elution volume is a trade off between DNA concentration and DNA recovery Lower volumes will give higher DNA concentrations but will sacrifice total DNA eluted Let the column stand for 1 minute before continuing Make sure to dispense the elution buffer directly on to the center of the spin column membrane taking care to avoid contact with the wall of the column or the membrane Spin at 13 000 RPM for 30 s to elute DNA from the spin column membrane Discard the spin column and save the 1 5 ml tube containing your sample The excised DNA fragment can be stored at 20 C in your personal freezer box or used immediately in downstream applications
9. trol switch on the top right hand side of the front panel to the left Put on orange goggles or use the orange plastic shield The LED blue light is very bright and will be uncomfortable if your eyes are exposed to it 2 Use plastic gel cutters to excise your band of interest and place it into a labelled 1 5mL microfuge tube Gel cutters can be reused please clean with a kimwipe 3 Weigh the small piece of agarose on scale inside of 1 5 mL microfuge tube 4 Add three volumes of ADB Buffer to 1 volume of agarose gel assuming that 100 mg of agarose gel is approximately 100 ul If using a 2 agarose gel use six volumes instead 5 Incubate 1 5 mL microfuge tube at 50 C until gel slice has completely dissolved 5 10 minutes Vortex the tube every 2 3 minutes to aid in dissolving the agarose 6 Optional For DNA fragments larger than 8 kb recovery can be improved by adding one additional volume volume of original gel slice of water to the mixture 7 Place Zymo Spin column in a 2 mL collection tube 8 Add the melted agarose mixture to the Zymo Spin column and spin at 13 000 RPM for 30 seconds Discard flow through and repeat as necessary until all mixture has been bound to the column ChE 130 Spring 2015 10 11 12 13 14 Wash DNA by adding 200 ul of DNA Wash Buffer to the Zymo Spin column and spin at 13 000 RPM for 30 seconds Repeat the wash repeat step 7 Remove spin column from collection tube and place in a
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