PCR Flashcards
What protein is stained purple in gram staining?
Meurine/ peptidoglycan
What is ethanols role in the gram stain?
Removers outer lipopolysaccharide membrane on gram and remove violet/iodine complex from peptidoglycan layer
What is the name of the complex that stains gram positive bacteria?
Counter staining to stain the gram bacteria
What are the four genetic techniques
Restriction enzyme
Gel electrophoresis
PCR
DNA sequencing
What do restriction enzymes do?
Cuts the DNA sequennces att a specific sight and produces DNA fragments with known sequences at the end.
What are sticky ends
Where the DNA sequence has been cut and known base pairs are exposed
What is a primer?
A strand of DNA which binds to the sticky ends and provides a sight for the polymerase to bind.
What does PCR do ?
It selectively amplifies a particular segment of DNA, this segment may represent a small part of a large and complex mixture.
What do you need to do PCR?
Starting material- Template DNA (original sample from a disease or person involved in crime)
Primers- up to 25 base pairs long, these attach to the template strand
Nucleotides- come along after the primer has attached and then attach to make it longer
Taq polymerase- Bacterial polymerase from a thermoally stable bacteria so will not denature at high temps
Salt Buffer- Keeps things balanced and neutral.
Stages of PCR
Denaturation- Seperates the template DNA from its double strand into its single strand and you have to heat it to 90 degrees
Annealing- Primers are attaching to the sticky ends of your fragment which you have created with the restriction enzyme and takes place in about (cooled down) 55 degrees
Elongation- Spare complementary neucleotides will come in and extend the strand.
Repeat step 3
What does PCR do?
Amplify sections of the DNA and amplify them rapidly.
What is PCR used for?
Disease screening
Species identification
DNA fingerprinting
DNA profilling steps
Obtain DNA sample
Create fragments of DNA
Amplify DNA- PCR
Seperate the fragments- gel electrophoresis
Visualise the fragments
Stain them and then compare them to a DNA ladder to get your results
Process of Gel Electrophoresis
Direct current is used which means the charge flows in one direction
DNA has a negative charge so it is attracted to the positive end of the tank because of an electrical current.
Then add a buffer to carry the charge
Smaller the size travel further faster and vice versa
How to set up gel electrophoresis
- Fill the tank with buffer solution
- Insert the electrodes into the take
- Attach electrodes to the power supply
- Put in the gel (agarose)
- Load the DNA sample into the sample wells at the negative end
- Apply a charge/current
- DNA fragments will move towards the positive electrode
= size and charge - Stain the gel
- Compare it to the DNA ladder
