Intro
DNA can be very useful for many things varying from Crime to Paternity. DNA profiling, or fingerprinting can be used to analyze suspects, but it can also be used to solve an uncertain pregnancy. A method called Restriction Fragment Length Polymorphism (RFLP) is currently preferred in the forensic world. RFLP provides a banding pattern on Restriction Sites on the individuals DNA. Restriction enzymes are a natural, bacterial defense mechanism. A Restriction Enzyme is like a small pair of molecular scissors. Restriction Enzymes destroy DNA from invading viruses or bacteriaphages. Restriction enzyems are used to cut DNA strands in Agarose gel electrophoresis. Agarose gel electrophoresis separates DNA fragments by size. DNA is negatively charged, so it will be attracted to the positive anode at the other end of the gel. Since everyone has different DNA, scientists have to create a way to locate, identify, and compare DNA of different individuals. Scientists probe the DNA so that they can identify a particular strand of DNA. This is called a "Radioactive Tag". The tag can also be used to demonstrate genotype similarites between individuals. The size of the fragments reflect variations in individual's DNA. Evidence for DNA fingerprinting can be collected from body tissues, bodily fluids(blood semen), hair follicles, etc.
Procedure
PT 1.
1. Label 6 micro test tubes CS, S1-S5
2. Pipette 10 microliters of each DNA sample into the micro test tubes
3. Pipette 10 microliters of Enzyme Mix into the bottom of each tube
4. Microcentrifuge the tubes to both mix the two substances and make them all at the bottom of the micro test tube.
5. Place tubes into large volume of water heated to 37 degrees C.
PT 2.
1. Centrifuge micro test tubes to bring all liquid to the bottom
2. Add 5 microliters of loading dye to each micro test tube.
3. Remove Agarose Gel and plastic wrap
4. Place Agarose gel into electrophoresis apparatus. Fill electrophoresis chamber with 1x TAE buffer to cover the gel, using approx. 275 mL of buffer for Bio-Rad mini-Sub Cell, horizontal electrophoresis chamber.
5. Using separate tip for each tube, load 10 microliters from the M tube, and 20 microliters from all of the other tubes. (CS, S1-S5).
6. Place lid back onto electrophoresis chamber, hook up the correct jacks with the correct colors (Red to Red, Black to Black).
7. Turn on power to 200V for 12 minutes.
8. Stain gel and record results.
DONE!
Results/Observations
Procedure
PT 1.
1. Label 6 micro test tubes CS, S1-S5
2. Pipette 10 microliters of each DNA sample into the micro test tubes
3. Pipette 10 microliters of Enzyme Mix into the bottom of each tube
4. Microcentrifuge the tubes to both mix the two substances and make them all at the bottom of the micro test tube.
5. Place tubes into large volume of water heated to 37 degrees C.
PT 2.
1. Centrifuge micro test tubes to bring all liquid to the bottom
2. Add 5 microliters of loading dye to each micro test tube.
3. Remove Agarose Gel and plastic wrap
4. Place Agarose gel into electrophoresis apparatus. Fill electrophoresis chamber with 1x TAE buffer to cover the gel, using approx. 275 mL of buffer for Bio-Rad mini-Sub Cell, horizontal electrophoresis chamber.
5. Using separate tip for each tube, load 10 microliters from the M tube, and 20 microliters from all of the other tubes. (CS, S1-S5).
6. Place lid back onto electrophoresis chamber, hook up the correct jacks with the correct colors (Red to Red, Black to Black).
7. Turn on power to 200V for 12 minutes.
8. Stain gel and record results.
DONE!
Results/Observations
A
ReplyDeletemention your controls, variables, and predictions
By the way, can you add "P" for "Pallarino" to your name on the blab, so I can recognize which Mike you are? Thanks!