LAB 6 Gel Electrophoresis of DNA

I DNA + or -?

-

Materials used to prepare gel

-1X Trix-acetate-EDTA (diluting 50x stock solution) -Gel apparatus -1 g agarose and 99 ml of 1X TAE (we used 1.5g and 150 ml) -Ethidium Bromide

Samples used

-1kb ladder -total chromosomal DNA of E.coli -EcoRI restricted plasmid dna -PsTI restricted plasmid dna (one more but we didn't have it)

What did we learn in this lab?

-how to prepare an agarose gel -how to load DNA samples into slots -how to run an agarose gel electrophoresis -how to stain DNA in gel using Ethidium Bromide -how to visualize and document agarose gels with DNA bands

wells used

-ladder, total chromosomal, plasmid dna

What gel concentration did we prepare?

1%

Steps

1) Assemble apparatus 2) Add 1.5 g of agarose and 150 ml of 1X TAE 3) Mix well and place flask in microwave for 2-3 minutes 4) Carefully pour the gel onto the plate until the gel reaches 4-5 mm in thickness. Try to avoid bubbles as you pour the gel. 5) Insert the comb into the slots at the top of the casting tray. 6)Allow the gel to solidify (~15-20 min.). 7) Pick up the casting tray and turn it 90 degrees so that the tray is running in the direction of the length of the unit. 8)Pour the 1X TAE electrophoresis buffer into both chambers of the unit. Continue to pour until the buffer covers the surface of the gel by about 2 mm. 9) Allow the gel to sit in the 1X TAE buffer for about 5 min. Now slowly, evenly, and gently pull up the comb to remove it from the gel.

How to load DNA samples into gel slots:

1) Obtain tubes and label them 2) A, B, C, D, E 3) Ass 5 ul of loading dye to each of the tubes. 4) When loading just break the surface of the buffer over the well with the micropipette tip and slowly inject the sample. The sample should sink into the well. 5) Use the micropipette to carefully load 20uL of each sample into the appropriate well. Make sure that there is no air between the tip and the sample.

Steps in DNA Analysis

1. Isolate and Purify DNA 2. Cut DNA into fragments w/Restricted Enzymes 3. Perform Gel Electrophoresis to separate DNA fragments by size 4. Produce Autoradiograph to analyze DNA bands DNA analysis first of all includes physical mapping and Southern hybridization.

base pairs in 1 kb ladder

13 strands, 250bp-10,00 bp

How much time is gel left to solidify ?

15 to 20 min

Describe the comb

1mm thick with 15 teeth

What does the solid agarose act as?

A matrix: forms a tri-dimension net with pores comparable to the sizes of DNA fragments from 100 base pairs (bp) to several thousand base pairs or kilobases (kb).

How to run an agarose gel electrophoresis:

A) After all samples are loaded attach the negative and positive electrodes on the power supply B) Power to 110 V C) Run the gel until the dye has moved about 10 cm (45 min)

Gel electrophoresis

An alternative way to asses DNA concentration by measuring the fluorescence intensity of dyes that bind to nucleic acids and selectively fluoresce when bound. Procedure used to separate and analyze DNA fragments by placing a mixture of DNA fragments at one end of a porous gel and applying an electrical voltage to the gel

Loading dye used

Bromphenol Blue. This contains glycerol which weights the sample so that is sinks into the well during loading.

How does electrophoresis separate fragments?

By size

Why shiuldnt we flood the gel with buffer?

DO NOT FLOOD THE GEL WITH THE BUFFER AS THIS WILL SHORT CIRCUIT THE ELECTROPHORESIS AND INCREASE THE TIME OF THE RU

What happens if you pull the comb quickly?

DO NOT PULL ON THE COMB TOO QUICKLY AS THIS WILL CREATE A SUCTION THAT WILL BREAK THE WELLS

High voltage means:

Gel overheats and agarose melts

How to load samples:

Just break the surface of the buffer over the well with the micropipette tip and slowly inject the sample.

What side does the comb go?

Negative electrode.

How to visualize the document agarose gels with DNA bands:

Photograph gel using a gel-doc system with camera for UV photography

Why DNA mapping?

Physical map (or restriction map) of the gene is created to determine which restriction enzymes cut the cloned gene, where and how many times.

Purpose of electrophoresis

Separate fragments

What causes the sample to sink to the bottom of the well?

The density of the dye

True or false: A level gel is necessary for the proper migration of the DNA samples during electrophoresis.

True

DNA Analysis

Used when researchers want to determine where a specific gene is located in a certain piece of DNA

Same intensity of longer and shorter fragments means?

We have two shorter fragments with similar sizes

result: plasmid with PstI

cuts at 2 sites two fragments with sizes 2.94 kb and 0.36 kb

result: double digestion

cuts at 3 sites 2.55kb 0.36kb 0.39kb

results: plasmid with EcoRI

cuts at one site linear dna fragment with size 3.3 kb.

DNA analysis is used to:

determine where a specific gene is located in a certain piece of DNA includes: -physical mapping -Southern hybridization

Low voltage means:

electrophoresis will go slower

results: total chromosomal with EcoRI

large number of restriction fragments with different sizes.

The larger the DNA fragments, the__________ it mill move through the gel

slower

Why is a physical map created?

to determine which restriction enzymes cut the cloned gene, where and how many times

What is the puspose of the loading dye?

used to track the progress of the sample