Lab: Molecular Bio Techniques For ID Of Orgs Flashcards
How is separation achieved?
Placing sample into porous media that allow molecules to move based on size
Gel electrophoresis is a technique that
Is used to separate molecules; can be used to separate nucleus acids and proteins
Media used for separation is
Agarose
Gel rig produces an electric field which
Passes through the gel and causes the DNA molecules to move through the gel according the charge and size
DNA has a net _____ charge, causing it to
Negative, move towards positive electrode
Smaller DNA fragments appear
Near bottom of gel
DNA ladder
Composed of DNA fragments that are varying but known size
Why use dye when loading the sample?
Allows for visualization and glycerol binds to DNA making it heavier than buffer
Ethidium bromide (etbr)
Fluorescent chemical added to agarose gel
Mutagen that disrupts and mutates DNA
Positively charged intercalating agent that sticks to DNA
Fluoresces a pink color
Where should band be for 16s rRNA?
1500bp
PCR is a method used to…
Amplify a specific region of DNA
Components needed to amplify a specific region of DNA
- reaction buffer to maintain pH
- oligonucelotide primers with specific sequence that anneal template strand and proved free 3’OH
- forward and reverse primers
- Taq polymerase (thermal stable)
- deoxynucleotide triphosphates (dNTPs)
- Mg2+ (cofactor for Taq)
Phases of PCR
- 95 DNA denatures –> single-stranded
- 50-65 allow forward and reverse primers to anneal to the targeted region
- 72 DNA polymerase binds to free 3’OH
- synthesis continues until temp increased to 95 or runs out of template
How many times is the cycle of PCR repeated?
29 times, results in exponential increase
What targeted sequence is used for PCR?
16s rRNA
