Lab 4: Epigenetics


Objectives

• Check success of amplification by running PCR product from Lab 3 on agarose gel
• Measure cytosine methylation using dot blot and chromogenic immunodection methods

Agarose Gel Electrophoresis
Last week we performed a PCR in class using your freshly reverse transcribed cDNA samples as template and primers for one of four genes. This week we will be checking if amplification was successful using electrophoresis and the agarose gel made during lab.


Procedure Background

• Nucleic acid molecules are separated by applying an electric field to move the negatively charged molecules through an agarose matrix. Shorter molecules move faster and migrate farther than longer ones because shorter molecules migrate more easily through the pores of the gel. This phenomenon is called sieving.
• The most common dye used to make DNA or RNA bands visible for agarose gel electrophoresis is ethidium bromide usually abbreviated as EtBr. It fluoresces under UV light when intercalated into DNA (or RNA). By running DNA through an EtBr-treated gel and visualizing it with UV light, any band containing more than ~20 ng DNA becomes distinctly visible. EtBr is a known mutagen, however, so safer alternatives are available.
• A DNA ladder is a solution of DNA molecules of different lengths used in agarose gel electrophoresis. It is applied to an agarose gel as a reference to estimate the size of unknown DNA molecules
• If amplification was successful you should see one clear band between 150-400bp (in length depending on your gene) and the negative controls will have no band. If there is a band in the negative control than there might be contamination in your reagents and you can not be sure that the intended gene was actually amplified.
• Often contamination requires carful rePCR in case the contamination occurred during reaction setup. However, if any reagents are contaminated troubleshooting may be required to obtain a clean PCR product.
• PCR bands outside of the intended size range could indicate unspecific amplification and will require either optimization of the reaction cocktail and thermal cycling parameters or redesigning the primers.

ELECTROPHORESIS PROCEDURE

1. Place gel in gel box and fill with 1x TAE buffer (to fully cover wells)
2. Remove combs from wells
3. Load 7uL 100bp ladder in far left lane
4. Load 25uL of your PCR sample into the gel (retain the remaining vol at -20ºC)
5. Run gel at ~ 100V for ~ 1hr
6. Visualize the gel on the UV transilluminator

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Cytosine Methylation Dot Blot


Procedure Background

• There are three primary components to this procedure: making dilutions of your DNA sample, binding your DNA sample dilutions to a nylon membranes (Dot Blotting), chromogenic Immunodetection of methylated cytosines using the Western Breeze kit and a 5meC antibody
• DNA is applied directly on a membrane as a dot using a vacuum manifold. This is then followed by detection of methylated cytosines using a 5-MeC antibody (Antibody product info). For dot blotting we will be using a protocol adapted from Schleicher and Schuel and the Western Breeze manufacture’s protocol for chromogenic innumodetection.

Chromogenic innumodetection consists of the following primary steps:

• Blocking- The membrane is blocked in order to reduce non-specific molecule interactions between the membrane and the antibody. This is achieved by placing the membrane in a solution of non-specific proteins (usually BSA or non-fat milk). The proteins in the blocking solution coat the remaining areas of the membrane where no protein is bound from the transfer. The reason this is necessary is described in the next step.
• Primary Antibody hybridization- The membrane is incubated with the first antibody. The primary antibody is specific for the molecule of interest, which in this case is methylated cytosines, and at appropriate concentrations, should not bind any of the other molecules on the membrane. Remember, antibodies are molecules, too. If we had not blocked the membrane, the antibody would end up binding to both the membrane and your target molecule. This would result in extremely high background (signals not related to the intended target protein(s)) and would use up a significant amount of the available antibody, making the interpretion of results difficult, if not impossible.
• Secondary Antibody hybridization- After rinsing the membrane to remove unbound primary antibody, a secondary antibody is incubated with the membrane. It binds to a species-specific portion of the primary antibody. Due to its targeting properties, the secondary antibody tends to be referred to as "anti-mouse," "anti-goat," etc., depending on the animal species that the primary antibody was created in. This secondary antibody is typically linked to an enzyme that allows for visual identification. In our case, the antibody is linked to an alkaline phosphatase (AP).
• Developing-The unbound secondary antibodies are washed away, and the enzyme substrate is incubated with the membrane so that the positions of membrane-bound secondary antibodies will either change color or emit light. Band densities in different lanes can be compared providing information on relative abundance of the protein of interest. The kit we will be using for visualization (Invitrogen's WesternBreeze Chromogenic Kit) utilizes an enzymatic reaction that creates a dark purple precipitate that can be seen with the naked eye.
• The chromogenic system emplyed in the WesternBreeze Chromogenic Kit is the combination of BCIP (5-Bromo-4-Chlo ro-3'-Indolypho sphate p-Toluidine Salt) and NBT (Nitro-Blue Tetrazolium Chloride). Together they yield an intense, insoluble black-purple precipitate when reacted with Alkaline Phosphatase.

METHYLATED CYTOSINE DOT BLOT PROCEDURE

DNA DILUTIONS

1. Sign-up for a DNA sample and note the type of DNA you have chosen
2. Label five snap cap 1.5 ml tubes with your initials, “DNA”, and the appropriate target concentration
3. Prepare five dilutions of your DNA, one of each target concentration, using the table provided. You should have 200ul of each dilution.
4. Set DNA aside to observe set-up dot blot vacuum manifold

Dilution	TARGET concentration	ul of H20	ul of 20X SSC	ul of 50ng/ul DNA sample
1	0.8 ng/ul	124	60	16
2	0.4 ng/ul	132	60	8
3	0.2 ng/ul	136	60	4
4	0.1 ng/ul	138	60	2
5	0.05 ng/ul	139	60	1

DOT BLOTTING

1. Cut nylon membrane to fit 72 wells of manifold
2. Soak nylon membrane in 6X SSC (enough to cover) for 10 min in top of tip box
3. Cut filter paper to size of the nylon membrane and wet in 6X SSC
4. Assemble manifold with the membrane lying on top of the filter paper
5. Denature DNA in boiling water for 10 m. Immediately transfer to ice
6. Switch on vacuum. Apply 500ul of 6X SSC to each well and allow SSC to filter through.
7. Spin down DNA for 5 min
8. Apply entire volume of DNA to wells. Be carful not to touch the membrane.
9. Note where you have applied your samples and each samples concentration on the blot map
10. Allow samples to filter through
11. While samples are being pulled through nylon membrane, soak filter paper cut to size in denaturation buffer
12. Once filtered through, dismantle manifold and transfer membrane to filter paper soaked in denaturation buffer and let sit for 5m
13. Place membranes on dry filter paper and let dry
14. Wrap dryed blot in plastic wrap and place DNA-side-down on UV transluminator for 2 mins at 120kJ. This immobilizes the DNA

WesternBreeze® CHROMOGENIC IMMUNODETECTION

General Guidelines

• Avoid touching the working surface of the membrane, even with gloves. Use forceps
• Work quickly when changing solutions as membranes dry quickly.
• Add solutions to the trays slowly, at the membrane edge, to avoid bubbles forming under the membrane. Decant from the same corner of the dish to ensure complete removal of previous solutions.

1. Prepare 20 mL of Blocking Solution

• a. Ultra filtered Water 14 ml
• b. Blocker/Diluent (Part A) 4 ml
• c. Blocker/Diluent (Part B) 2 ml
• d. Total Volume 20 ml

2. Place the membrane in 10 ml of Blocking Solution in a covered, plastic dish.
3. Incubate for 30 minutes on a rotary shaker set at 1 revolution/sec.
4. Decant the Blocking Solution.
5. Rinse the membrane with 20 ml of water for 5 minutes, then decant. Repeat.
6. Prepare 10 mL of Primary Antibody Solution (1:5000 dilution)

• a. Blocking Solution 10 ml
• b. 5-MeC antibody 2 µl
• c. Total Volume 10 ml

7. Incubate the membrane with 10 ml of Primary Antibody Solution for 1 hour. DURING THIS INCUBATION PERFORM AGAROSE GEL ELECTROPHORESIS
8. Decant primary antibody and wash the membrane for 5 mins with 20 ml of TBS-T. Decant and repeat three more times
9. Incubate membrane in 10 ml of secondary antibody solution for 30 mins. Decant
10. Wash the membrane for 5 mins with 20 ml of TBS-T. Decant and repeat three more times
11. Rinse the membrane with 20 ml of water for 2 minutes, then decant. Repeat twice.
12. Incubate the membrane in 5 ml of Chromogenic Substrate until color begins to develop (1-60 mins)
13. Rinse the membrane with 20 ml of water for 2 minutes, then decant. Repeat twice.
14. Dry membrane of clean piece of filter paper



We will go over how to analyze results in lab next week