Lab: Molecular Bio Techniques For ID Of Orgs Flashcards Preview

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Flashcards in Lab: Molecular Bio Techniques For ID Of Orgs Deck (19):
0

Gel electrophoresis is a technique that

Is used to separate molecules; can be used to separate nucleus acids and proteins

1

How is separation achieved?

Placing sample into porous media that allow molecules to move based on size

2

Media used for separation is

Agarose

3

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

4

DNA has a net _____ charge, causing it to

Negative, move towards positive electrode

5

Smaller DNA fragments appear

Near bottom of gel

6

DNA ladder

Composed of DNA fragments that are varying but known size

7

Why use dye when loading the sample?

Allows for visualization and glycerol binds to DNA making it heavier than buffer

8

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

9

Where should band be for 16s rRNA?

1500bp

10

PCR is a method used to...

Amplify a specific region of DNA

11

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)

12

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

13

How many times is the cycle of PCR repeated?

29 times, results in exponential increase

14

What targeted sequence is used for PCR?

16s rRNA

15

What is DNA purification?

Used to remove all unnecessary components from the PCR reaction except for the PCR product

16

The montage system

- relies on filtration
- uses membrane filter to trap DNA
- centrifuge nucleus acids into tube

17

The ExoSAP-IT system

- uses enzymatic cleanup of DNA
- exonuclease I removes residual single-stranded primers
- shrimp alkaline phosphatase removes remaining dNTPs from reaction

18

What are the universal primers?

F27
R1492