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Pre-Lab Topics

• Agarose gel electrophoresis
• Restriction enzymes

Experimental Procedures

• RNA isolation
• Agarose gel analysis of RNA

Overview of Procedures

You and your partner will isolate RNA from Arabidopsis.  To confirm the quality of the isolated RNA, you will a) measure absorbance at 260 and 280 nm and determine the ratio and b) analyse RNA on an agarose gel.

Notes on Molecular Biological Procedures

General Guidelines
1. Maintain a clean work area
2. Use a fresh pipet tip for every transfer (tips should be DNase/RNase free)
3. Wear gloves to prevent contamination (of yourself as well as your experiment)
4. Sterilize solids and liquids by autoclaving 20 minutes at 121°C at 15 psi

Pipetting Small Volumes
1. Before beginning the procedure, thaw all frozen reagents and mix well
2. Pulse spin ALL tubes of aliquots to bring the liquid to the bottom of the tube -- in the microcentrifuge hold the "SHORT" key for about 5-10 seconds
   (Capless 1.5 ml vials serve as holders for 0.2 and 0.5 ml tubes in the microcentrifuge rotors)
3. Touch only the very tip to the surface of the solution (i.e., do NOT submerge the pipet tip into the solution)
4. Most enzyme stocks are in 50% glycerol; these solutions are quite viscous and liquid will stick to the outside of the pipet tip so touch only the surface

Centrifugation
1. DO NOT PUT TAPE ON TUBES!
2. ALWAYS balance the load in the centrifuge
3. Capless 1.5 ml vials serve as holders for 0.2 and 0.5 ml tubes in the rotors
4. Pulse spin ALL tubes of aliquots to bring the liquid to the bottom of the tube -- in the microcentrifuge hold the "SHORT" key for about 5-10 seconds
5. DO NOT SLAM THE LIDS! (this action breaks the latch mechanisms)

Agarose and Acrylamide Gel Electrophoresis of DNA and RNA

In both gel media, DNA is driven through the matrix by electric current. Smaller or more compact molecules pass through the matrix easier and migrate farther than large molecules. All linear DNA has the same charge per unit length and linear pieces migrate according to size. Plasmid DNA preparations contain three types of DNA conformations: linear, relaxed circular (or nicked) and supercoiled. Only the linear form can be used to estimate the size of the molecule. Usually, but not always, the supercoiled runs fastest, linear next, then the relaxed circular. A carefully prepared sample will be mostly supercoiled. The range of sizes separated in a gel is controlled by the % of agarose or %T of acrylamide in the gel.

Resolution Versus Matrix Concentration

The mobility is proportional to the voltage applied at low voltage but increasing voltage decreases the resolution of larger fragments of DNA. A general guideline for agarose gels in 1xTBE is 5V/cm maximum for resolving fragment lengths greater than 2 kb. The distance between the electrodes serves as the length in the calculation. Higher voltages increase the temperature of the gel causing increased band width and distortion of the lanes. The agarose can also melt, especially the low melting point agarose sometimes used when DNA is to be recovered from the gel. Voltages for acrylamide gels are generally twice the recommended voltage of the agarose gels but it is important to check manufacturer recommendations for the gel or for the electrophoretic equipment.

The mobility is also influenced by the choice of buffer systems. Besides the Tris Borate EDTA, pH 8.3 (TBE) buffer used in our experiments, a Tris Acetate EDTA buffer (TAE) is preferred by some. The TAE buffer shifts the range of resolution toward higher fragment lengths.

Denaturing gels can also be run to separate fragments as single stranded DNA. Gels can be run at high pH (30 mM NaOH) or in neutral buffers when glyoxal is added to the gel and the running buffer. Only the glyoxal system is suitable for RNA separations. Why is there a restriction of systems for RNA separations?

The nucleic acids are visualized with ethidium bromide (EtBr). This fluorescent dye, which contains a tricyclic planar group, intercalates between stacked base pairs of nucleotides and, in this environment, fluoresces when excited with ultraviolet light; the fixed position of the planar group and its close proximity to the bases causes dye bound to DNA to display increased fluorescent yield compared to free dye.

• UV radiation at 254 nm is absorbed by DNA and transmitted to the dye, whereas radiation at 302 and 366 nm is absorbed by the bound dye
• energy is re-emitted at 590 nm in the red-orange region of the visible spectrum
  
  NOTE: most commercial UV light sources emit at 302 nm, which yields slightly less fluorescence than at 254 nm but produces LESS nicking of DNA

The size of linear fragments of DNA is determined by comparison to standards in the same manner as protein molecular mass is determined from SDS-PAGE. The log (# base pairs) is plotted versus distance migrated or Rf value {Helling R.B., Goodman H.M., and Boyer H.W. 1974. Analysis of endonuclease R-EcoRI fragments of DNA from lambdoid bacteriophages and other viruses by agarose-gel electrophoresis. J. Virol.14: 1235-1244}.

The tracking dye combined with the DNA samples contains bromophenol blue and xylene cyanol for use in visually monitoring electrophoresis and glycerol to make the sample dense enough sink to the bottom of the well. The stock solution is designed to be diluted about six fold in the sample. Bromophenol blue runs about the same size as a linear double-stranded DNA molecule of 300 base pairs in length in 1X TBE on a gel of 1% agarose. In low percentage gels of 0.4% agarose, the dye can emulate a 1000bp fragment. Remember not to run this dye off the bottom of the gel when you are trying to analyze small fragments. Xylene cyanol runs about the same as a linear double-stranded DNA molecule of 4kb in a 1% agarose gel.

Isolation of Total RNA from Arabidopsis thaliana

• Total RNA will be isolated from Arabidopsis using ??
  
  
• The concentration of RNA will be determined by measuring the absorbance at 260 nm; A260/A280 will be determined to give an estimate of the purity of RNA with respect to protein contaminants.
  
  
• The integrity of the 26S and 18S ribosomal bands will be verified by agarose gel electrophoresis; the ribosomal bands should appear as sharp bands:
  
  
  *Sample agarose gel of RNA from Arabidopsis

Agarose gel electrophoresis of RNA

1. Sample Preparation
   
   NOTE: 6X loading buffer III contains
   0.25% bromophenol blue
   0.25% xylene cyanol FF
   30% glycerol (in water)
   
   (from Sambrook, J., Fritsch, E.F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, Second Edition (Cold Spring Harbor Laboratory Press))
   
   
   • Standards:
     Quick-Load® 1 kb DNA Ladder (New England Biolabs, Ipswich, MA): load 10 µl (0.5 µg) per lane
     *Record this information in your notebook and include both sizes (in bp) and mass of ladder fragments.
     **This ladder can be used to quantitate the amount of DNA in a sample; the mass of DNA in each band in the ladder has been calibrated (the 3,001 bp fragment has increased intensity to serve as a reference band).
     
     
   • PCR:
     a. Spot 1 µl 6X loading buffer (LB) on a piece of parafilm (one spot for each PCR sample).
     b. Using a clean pipet tip, make a hole in the center of the paraffin wax overlay; use a fresh tip for each tube.
     c. Insert a fresh tip through the hole and remove 5 µl from the 1st PCR tube, mix it with one spot of 6X LB by pipetting up and down, and load the sample into the well.
     d. Repeat procedure for each PCR reaction.
   
   
2. Run the gel at 130 V for 30-40 minutes (check gel at ~ 20 min). Refer to protocol for agarose gel electrophoresis.
   
   
   Expected products:
   • Positive control (Promega) = 323 bp
   • Negative control = NO bands
   • Upstream regulatory sequence = ??
   
   
3. IF PCR worked, take pictures (each team should bring a digital camera to lab); compare the size of the PCR product against the 1 kb ladder.
4. Store gel at 4°C and rinse out the gel apparatus with RO water.
   
   
   • if PCR worked, choose the reaction with the higher yield and proceed with DNA Clean-Up
   • if BOTH reactions worked, give us the extra product for "backup" PCR
   • if PCR did NOT work, get a positive PCR sample from us and proceed with DNA Clean-Up

Alternative Gel Documentation with the UVP BioDoc-It™ System: this option will be available if you do not have a digital camera; the image is printed on thermal paper and is in black/white

System Components

• CCD Video Camera
• Zoom Lens
• Transilluminator (302 nm)
• UV/White Light Converter Plate
• Darkroom Cabinet
• LCD Monitor
• Thermal Printer

1. Position the gel in the center of the plate on plastic wrap.
2. Close the darkroom cabinet's door and turn on the transilluminator
   
   NOTE: the transilluminator will cut off if the darkroom door is opened.
   
   
3. Adjust the lens f-stop adjustment (TOP ring) to increase or decrease the brightness of the image.
4. Rotate the zoom adjustment (MIDDLE ring) on the lens so that the image is the appropriate size.
5. Press "Print" on the thermal printer in order to print the image; press AND hold copy/feed to advance the paper to cut the picture.
6. Carefully remove the gel from the plate; wipe off the plate with Kim wipes.

Copyright, Acknowledgements, and Intended Use
Created by B. Beason (bbeason@rice.edu), Rice University, 25 June 1999
Updated 20 July 2011