• Day 1
• Day 2
• Day 3
• Day 4
• Day 5
• Day 6

Resources

• *LAB SAFETY**
• Laboratory notebook
• Graphing tutorial
• Molecular biol tips
• Writing assignment
• Bios 211 web site
• Searching literature

Other links

• Bios 111 honor code
• Graphing quiz (rice only)

Bios 111 Day 2

Agarose Gel Analysis and Gel Purification of Plasmid DNA

Introduction

DNA work will continue with the following activities. Today you will use analyze the plasmid DNA that you prepared last time using agarose gel electrophoresis, purify the digested DNA from the gel, and inoculate your LB plates with an overnight bacterial culture.

Background

AGAROSE GEL ELECTROPHORESIS OF DNA

DNA is driven through the agarose matrix by electric current. Smaller or more compact molecules pass through the matrix easier and migrate farther than large molecules. All DNA has the same charge per unit length and linear pieces migrate according to size. The range of sizes separated in a gel is controlled by the % of agarose in the gel.

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.  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.

Ethidium Bromide

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.

If a DNA sample is too dilute to measure at 260 nm or is contaminated with other compounds that absorb in the UV range, the amount of DNA present can be estimated from the intensity of ethidium bromide fluorescence. Since the amount of DNA in a solution is proportional to the fluorescence emitted by ethidium bromide, the DNA quantity in an "unknown" solution can be estimated by comparing its level of fluorescence with the intensity of known amounts of DNA.

The molecular weight DNA markers used in our study are Quick-Load 1 kb DNA Ladder (New England Biolabs, Catalog #N0468S). The ladder (see figure) produces eleven DNA fragments ranging in size from 500 to 10,000 base pairs (see picture). 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 size of linear fragments of DNA is determined by comparison to standards: the log10 (# 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 loading buffer (LB) combined with the DNA samples contains two tracking dyes, bromophenol blue and xylene cyanol, for 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.

Experimental overview

Today’s procedures involve gel analysis and clean-up of digested DNA.  On some procedures you will work as an individual; on others you will work with a partner.  Perform the procedures in the order given below.  Make sure that you use the appropriate pipettor and set the volume correctly—if you’re unsure, then ask.  Record all procedures and data in your lab notebook, indicating “who” performed a procedure step when you work as pairs; turn in copies of notebook pages at the end of the laboratory session.

• Agarose gel electrophoresis of DNA (pair)
• Gel purification of digested DNA (pair)
• Streaking of plates to get individual colonies (individual)

SPECIAL NOTE:  Record enough procedure details in your notebook during lab today so that you can repeat these procedures using your notebook as the ONLY resource.  Write the methods in your own words (i.e., do not just “copy” the steps from the web page or handouts).

A) Agarose gel electrophoresis of DNA [1% agarose in 1X Tris-borate-EDTA (TBE)]

PROTOCOL

1. Thaw uncut DNA and PstI digest at room temperature
2. Pulse spin uncut and digested samples to bring contents to the bottom of the tube
3. Prepare samples (see below). For agarose gels it is advisable to load the same volume into each well. Some samples may need to be diluted with water or Tris-EDTA (TE) to achieve this.

“Uncut control” plasmid DNA: add 5 µl 6X loading buffer (LB)
“PstI-digested” plasmid DNA: add 5 µl 6X LB

4. The instructor or TA will load 10 µl of NEB’s Quick-Load 1 kb DNA ladder.
5. Carefully load ALL of each sample (30 µl) into different wells in the gel and record the order of the samples in your notebook (record the location of each sample, not just the ones that you loaded). Do not press the tip into the bottom of the well while loading--allow the sample to sink there.
6. Position the lid and connect the electrodes so that the anode is at the bottom of the gel (“run to red”).  PLEASE NOTE: Banana plug fittings are not to be turned or twisted. Only push on and pull off by grasping the plug (or the entire lid for the boxes) without turning.
7. Turn on the power supply (switch is located on the right side).
8. Select “voltage” and set the voltage to 130 V using the raise/lower arrow keys; after the voltage is set, press the RIGHT SELECT button until DISPLAY is lit to monitor the actual voltage (v) and current (ma) during the run.
9. Press RUN.  The 500 bp standard will run just behind the dark blue dye front, and smaller fragments that run ahead of the dye may not be visible in this type of analysis.

CAUTION: Lethal voltages are present while the power supply is "ON." Do not touch the gel or buffer until the electrodes are disconnected.

10. STOP the run after ~ 45 minutes.  Once the voltage is “zero” turn off the power supply and carefully remove the lid.
11. Wear gloves and place casting tray with gel onto paper towels and carefully carry to the photography area. DO NOT spread EtBr outside the designated area!! From this point forward, assume that your gloves are contaminated with EtBr. Do not touch anything with those gloves that is not supposed to be contaminated.
12. Place gel onto a sheet of plastic wrap on the transilluminator.  CAUTION: The gel is still laden with EtBr and should be handled only with gloved hands. Scrupulously avoid all skin contact with the gel.
13. The instructor or TA will take pictures for you using the UVP BioDoc-It™ System (components are listed below).
14. Proceed to B): After excising the desired DNA band, dispose of the gels in the Biohazard Waste Box.  Do NOT put paper towels, plastic wrap, or gloves in this waste box--ONLY the gels.

ATTENTION: Avoid doing anything that would unintentionally contaminate the transilluminator or camera with EtBr. For instance, do NOT lay gels directly on the transilluminator, but always on plastic wrap. Do NOT contaminate the equipment (door knob, camera, printer, etc.)--REMOVE your gloves FIRST.

Gel Documentation with the UVP BioDoc-It™ System

System Components

• CCD Video Camera
• Zoom Lens
• UV Blocking Filter--the orange-colored filter absorbs UV and IR radiation from the transilluminator and enhances the orange/pink bands of ethidium bromide stained gels
• Transilluminator (302 nm)
• Darkroom Cabinet
• LCD Monitor
• Thermal Printer

B) Gel purification of digested DNA (work as pairs and cut out ONE band of DNA)

The restriction enzyme must be removed before setting up the ligation reaction; any residual restriction enzyme could re-digest the plasmid, thus decreasing the likelihood of obtaining bacterial transformants.  One way to remove enzymes is agarose gel purification.  We’re using the Zymoclean Gel DNA Recovery Kit™ (Catalog No. D4001 & D4002, Zymo Research Corp.) to purify and concentrate the PstI-digested plasmid DNA. The desired DNA band is cut from the gel; the agarose is dissolved and the DNA is recovered using a spin column. The gel-purified DNA is suitable for DNA ligation reactions, DNA sequencing, PCR, etc.

PROTOCOL (from the Zymoclean Gel DNA Recovery Kit™ Instruction Manual)

1. Locate the digested vector DNA band on the agarose gel using long wave ( >300 nm) UV light.

   Wear a face shield to protect your eyes and minimize exposure time to skin.

2. Excise the DNA from the gel using a razor blade or scalpel (use a fresh one for each piece of DNA; dispose of in a sharps container).  Cut as small a piece of agarose as possible -- trim off excise gel around the band.
3. Transfer gel piece to a sterile 1.5 ml tube; label the tube
4. Pulse spin for ~ 10 sec to "pellet" the agarose (for estimation of gel volume)
5. Add 3 volumes of ADB Buffer™ to each volume of excised agarose
6. Incubate at 55°C (in a water bath) until the agarose is completely dissolved (5-10 minutes)
7. Add the melted agarose solution to a spin column in a collection tube
8. Centrifuge at 16,000 x g for 30 seconds and discard column flow-through (liquid waste)
9. Add 200 µl wash buffer (contains ethanol) and centrifuge as in step 8
10. Repeat the wash and centrifuge as in step 8; put column in a sterile 1.5 ml tube
11. Add 15 µl nuclease-free water to center of column
12. After 1 minute, centrifuge at 16,000 x g for 1 minute to elute DNA
13. Store gel purified DNA at -20°C in your Stratacooler

C) Streaking of plates to get single colonies

Growth and Check of Bacterial Strains

Bacteria can be propagated on liquid or solid media. The use of liquid allows large quantities of bacteria to be harvested but does not permit easy selection or determination of phenotype of single cells. The technique of "streaking" cells onto a solid media provides simple isolation of colonies arising from single cells. Colonies selected for the desired phenotype are then used to inoculate liquid broth. A single colony inoculum is preferred because bacteria can undergo many types of mutations naturally. The instability of some of the mutations, especially transposons and phages, can allow some cells to lose characteristics important to the selection scheme and may complicate the analysis. It is always wise to check the parent strain for proper phenotype that reflects the genotype and then use "picks" from single colonies to start liquid cultures.

Aseptic removal of toothpicks

PROTOCOL (dispose of toothpicks in biohazard bag, not in regular trash)

Single colonies can be produced in the following manner.

Touch a clean toothpick to the surface of the culture solution and lightly streak the culture onto surface of the plate (LB-kan) as indicated in the figure.  Many passes in a zigzag motion are recommended but do not overlap from line to line.
Remove a sterile toothpick from the tube and drag the end of the pick through a single portion of the preceding streak.  Continue a zigzag pattern on the plate being careful NOT to overlap with the previous streak.
Repeat the above step but drag across a single portion of the second streak as indicated in the figure.
Invert the plates and incubate at 37°C overnight.

Homework Assignments

Prepare all of the homework assignments in your laboratory notebook and turn in the duplicates at the beginning of the next laboratory session.

1. Use the picture of your gel from day 2 to hand draw a DNA standard curve in your notebook.  A computer-generated plot is not appropriate for this application. Plot log₁₀ (# base pairs) versus distance migrated from the well and use the graph to estimate the size of the PstI-digested plasmid DNA.  Show all measurements and calculations.
2. Compare the estimated size of the digested plasmid with the expected size (plasmid map).