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Honours test 1 > DNA extraction, electrophoresis and RE digests > Flashcards

DNA extraction, electrophoresis and RE digests Flashcards

0
Q

DNA extraction overview

A

Sample > add PBS > sucrose triton X-100 > TE buffer > SDS > proteinase K > incubate overnight > NaCl > alcohols > air dry

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1
Q

Three common methods for extracting DNA

A

Phenol-chloroform extraction
Silica based kits
Proteinase K and salting out

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2
Q

What protects bacterial DNA from restriction endonucleases

A

Methylation

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3
Q

Define isoschizomers

A

A pair of REs that recognize the same site and cleave at the same position

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4
Q

Define neoschizomers

A

REs that recognize same sight but cleave at different posititions

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5
Q

How do you ensure REs don’t get damaged

A

Work quickly
Work on ice
Add to mixture last

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6
Q

Factors affecting RE digests

A

Purity of DNA (protein, salt, RNA, solvents)
DNA methylation
Reaction conditions (pH, temp, ionic strength, concentration)
Enzyme to DNA ratio

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7
Q

How to improve DNA purity in RE digest

A

Purify DNA
Increase amount of RE
Increase reaction volume (dilute contaminants)
Increase incubation time

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8
Q

Possible causes of star activity

A 
High glycerol (>5%)
High enzyme:DNA ratio (>100units/ug)
Low ionic strength
High pH (>8)
Presence of organic solvents
Substitution of Mg with other divalent cations
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9
Q

Separation in gel electrophoresis based on

A

Size
Charge
Conformation

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10
Q

Types of gel electrophoresis

A 
Agarose - DNA
Formaldehyde - RNA
PAGE - DNA
SDSPAGE - protein
Pulsed field electrophoresis - large DNA
Capillary electrophoresis
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11
Q

What is agarose

A

Linear polysaccharide
Seaweed extract
Basic repeat unit = agarobiose

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12
Q

Role of electrophoresis buffer

A

Maintains pH
Provides ions for conductivity
(Tris acetate/tris borate)

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13
Q

Three purposes of loading buffer

A

Increase sample density (glycerol/sucrose/Ficoll)
Adds colour to sample
Contains dye to monitor progress of gel (bromophenol blue [linear ds 300bp] xylene cyanol [linear ds 4kb])

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14
Q

What is ethidium bromide

A

Planar molecule that intercalates between nucleic acid bases.
Fluoresces under UV light

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15
Q

Factors influencing DNA migration on gel

A

Molecular size of DNA
Agarose concentration
DNA conformation
Applied voltage

16
Q

Dense agarose gel separates …

Low conc agarose gel separates …

A

Smaller DNA fragments

Larger DNA fragments

17
Q

Mobility of super coiled DNA relative to linear depends on..

A

Agarose concentration
Strength of current
Ionic strength of buffer

18
Q

At high voltage ..

A

Large fragments run proportionally faster than smaller ones. (No more than 5V/cm)

19
Q

What is different about RNA gel

A 
RNA tends to form 2dry structures
Contain denaturants (formaldehyde, formamide)
All reagents/ apparatus treated with DEPC (diethyl pyrocarbonate) to remove all RNAses
20
Q

Difference between agarose and PAGE

A

Page has greater resolution but smaller range of fragments

PAGE (5-500bp) differentiate >1bp
Agarose (0.1-25kb) differentiate >20bp

21
Q

Define star activity

A

Relaxation or alteration of restriction enzyme mediated cleavage of DNA. Results in cleavage at non-canonical recognition sites.

22
Q

Difference between TAE and TBE

A

TAE - higher electric current = more heat = poor gel banding pattern. Good for short gel run times. LOWER buffering capacity

TBE - high resolution of DNA fragments, not recommended if wanting to extract/purify DNA. HIGH buffering capacity.

23
Q

How is PAGE gel formed

A

Acrylamide monomer forms chains in presence of free radicals
Chains become cross linked
Process stabilized by TEMED and inhibited by oxygen.

NOTE acrylamide is a neurotoxin

24
Q

What are non-denaturing PAGE gels used for

A

Separating and purifying dsDNA fragments.

Detecting small nucleotide differences.

25
Q

What are denaturing PAGE gels used for

A

Separating and purifying ssDNA frags
Agent (eg urea or formamide) prevents base pairing
Downstream - sequence analysis, genotyping, isolate radiolabelled probes.

26
Q

How to visualize DNA in PAGE

A 
Radioactivity (32P, dried under heat and vacuum, x-ray film)
Silver staining (silver solution binds DNA, reacts with formaldehyde in alkali - looks brown on yellow)

Honours test 1 (6 decks)

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