Presentation on theme: "Gel electrophoresis. +- Power DNA How fast will the DNA migrate? strength of the electrical field, buffer, density of agarose gel… Size of the DNA! *Small."— Presentation transcript:
+- Power DNA How fast will the DNA migrate? strength of the electrical field, buffer, density of agarose gel… Size of the DNA! *Small DNA move faster than large DNA …gel electrophoresis separates DNA according to size small large Within an agarose gel, linear DNA migrate inversely proportional to the log10 of their molecular weight.
Vertical Gel Electrophoresis for Fish Gel Fill chamber with buffer Break off wells Remove comb
I.A. Calculate the R f OF PROTEINS R f = migration distance of protein (origin to band) migration distance of dye front (origin to dye front) Figure 2. Formula for calculating the retardation factor (Rf) of a protein in SDS- PAGE. 1. Calculate the R f value for each protein band in the protein standard.
I.A. R f OF PROTEINS 2. Plot the molecular weights of your standard proteins (vertical axis) versus the R f values (horizontal axis) on semi-log graph paper.
I.A. R f OF PROTEINS 3. Draw a best-fit straight line through all your points. Molecular wts. R f value
I.A. R f OF PROTEINS 4. For each lane of fish protein extract, pick a single protein band and circle it on the graphic image that you will download from the class Laulima site. For each of these bands, determine its molecular weight using this standard curve.
Attempt to construct an evolutionary tree illustrating these relationships. I.B. PROTEIN FINGERPRINTING Hypothesize Evolution Relationships Among the Fish Species Analyzed
1. Lay out a small piece of Parafilm on a flat countertop. 2. Place a 1 μL drop of SYBR Gold dye (labeled "SG"; stains the DNA with a fluorescent dye) on the Parafilm. 3. Next to this drop, place a 1.7 μL drop of sample buffer (labeled "SB"). 4. Next to these drops, place a 2.3 μL drop of sterile water. 5. Next to these drops place a 5 μL drop of your DNA sample and mix, using your pipetter all four drops together. 6. Take up the entire mixture (10 μL) into a pipetter and carefully transfer to one of the wells in the agarose gel. Be sure to note which well your sample was applied to in your laboratory notebook. Loading the Gel Ladder:
7. Repeat these steps (1-6) with your PCR product, loading a different lane in the agarose gel. 8. The instructor will load a DNA ladder (allows molecular weight estimation of your DNA sample). 9. Once all of the wells are loaded, we will run the gel at 100 volts for about one hour. 10. After one hour has elapsed, we will visualize the gel under UV light. 11. Examine the gel image and identify which lanes contains your original DNA extract and your PCR product. Comparing the migration of your product to the DNA ladder, estimate the molecular weight of this product. Loading the Gel
buffer Add enough electrophoresis buffer to cover the gel to a depth of at least 1 mm. Make sure each well is filled with buffer. Cathode (negative) Anode (positive) DNA
6X Loading Buffer: SYBR Gold Dye (for color) Sample Buffer (SB) Sample Preparation Mix the samples of DNA with the 6X sample loading buffer (w/ tracking dye). This allows the samples to be seen when loading onto the gel, and increases the density of the samples, causing them to sink into the gel wells.
Loading the Gel Carefully place the pipette tip over a well and gently expel the sample. The sample should sink into the well. Be careful not to puncture the gel with the pipette tip.
Place the cover on the electrophoresis chamber, connecting the electrical leads. Connect the electrical leads to the power supply. Be sure the leads are attached correctly - DNA migrates toward the anode (red). When the power is turned on, bubbles should form on the electrodes in the electrophoresis chamber. Running the Gel (red +) (black -)
wells Bromophenol Blue Cathode (-) Anode (+) Gel After the current is applied, make sure the Gel is running in the correct direction. Bromophenol blue will run in the same direction as the DNA. DNA (-)
Nanodrop spectrometer used to measure DNA concentrations II.B. DNA Quantitation
Clean with 1 μl nanopure water Close, open Wipe with kimwipe. Blank 1 μl mastermix Press blank button Clean with kimwipe MEASURE click left Blank should read 0 Sample 1 μl DNA sample Nanodrop spectrometer READINGS Concentration Absorbance 260 nm 280 nm Purity Good purity if below 2 and above 1.8