DNA Techniques Lab Preparation

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Manipulating GenesGenetic Engineering: You can repair genes, insert genes, excise genes or replace genes with gene therapy

Manipulating DNA You can identify who was at a crime scene, a John Doe, paternity of children sequence DNA and more with DNA Technology

What will we do?We are going to identify who was at the crime scene of a vicious murder of Spongebob Squarepants

How do we figure out Who was at the crime scene?

Small amounts of trace evidence will be collected

In this case, body fluid was found near the body and collected

PCR Polymerase chain reaction Sometimes only tiny

amounts of evidence are found

PCR is like a photocopy machine for DNA, so lots of copies are made

What now? The DNA is cut into tiny pieces by

chemical scissors known as restriction enzymes (RE)

Each enzyme cuts at a specific sequence

Each person’s DNA is different, so each RE cuts everybody’s DNA into different numbers and sizes

Restriction EnzymesThere are lots of restriction enzymes that cut at different sequences

Gene InsertionMatch the overlapping ends with the appropriate bp and you can insert any gene you want!!!

The DNA for this lab has been pre-cut by restriction enzymes

WHAT NOW? Because everybody’s DNA is different, determining the sizes of each fragment is as good as a fingerprint!!!

How Do We Determine the size of the fragments?

Gel Electrophoresis separates the DNA according to size from biggest to smallest with the use of electricity!!!

What the heck is gel electrophoresis?

DNA is negatively chargedGel electrophoresis takes advantage of that property.

We will actually apply an electric current to the DNA

AGAROSE A semisolid material that has many purposes in science.

It is comparable to a sponge (little holes and big holes)

A comb makes wells

Fragmented DNA is inserted into the wells

Fragment size and agarose

Because agar has holes similarly sized fragments will get stuck at about the same places in the agar.

This allows us to actually see bands of DNA in the agar

Fragment size and agarose

If you have big pieces of DNA and little pieces of DNA which do you think would travel the fastest through the agarose?

The smaller pieces!!!

These are known as DNA fingerprints!!!

How do we set up the gel/tray?

DYE LAB – TEST RUN FOR GEL ELECTROPHORESIS

What did you do well on this lab?How could you improve your skills and how

could you reduce the time it took to set up the lab?

M=“Marker”(also known as DNA Size Standard – sample of digested DNA with known sizes of fragments

Get DNA samples Add 5 l of Loading Dye (LD) to each sample

BE SURE TO MIX IN MICROCENTRIFUGE!

Put 15 l of sample in each well

2 3 4 5M CS 1

Interpreting DNA Fingerprint Results

Rinse your gel and then view on

light box.

Ladder/Standard/MarkerIf we measure how far each set of fragments moved, we can graph the distance (in mm) VS the known fragment sizes (in base pairs) and use this as a kind of molecular ruler

Sample

Analysis of DNA Fingerprint Data

Use your metric ruler to measure distance each band traveled

Graph the DNA size standard

(AKA “standard marker”) distances

VSthe given size of the fragments in each band from

your table

Graph the DNA size standard

(AKA “standard marker”) distances

Graph the DNA size standard

(AKA “standard marker”) distances

Who was at the crime scene?

Paternity Testing

Graph the DNA size standard

(AKA “standard marker”) distances