Purifying DNA & RNA Source Amounts & Purity Damage or Loss

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1 Purifying DNA & RNA Source Amounts & Purity Damage or Loss

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Purifying DNA & RNA Source Amounts & Purity Damage or Loss. Cells. Extract. Remove junk. Pure DNA. Bind DNA. 1. Remove high MW junk. DNA, RNA Solution. Denatured Protein. Phenol. Cell Extract. Shake. Spin. 2. Remove low MW junk and concentrate. Add. (to 70%). and salt. - PowerPoint PPT Presentation

Transcript of Purifying DNA & RNA Source Amounts & Purity Damage or Loss

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Purifying DNA & RNA

Source

Amounts & Purity

Damage or Loss

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Bind DNA

Cells

Pure DNA

Extract

Remove junk

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DNA, RNASolution

DenaturedProtein

Phenol

CellExtract

Shake Spin

1. Remove high MW junk

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(to 70%)

SPIN (fast)

Add

and salt

2. Remove low MW junkand concentrate

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BIND + SALT(aided by alcohol)

ELUTE - NO SALT(just add water)

Or Bind DNA

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EXTRACT INHigh Salt

SILICAPARTICLES

WATER

DNA

High SaltWash

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Purifying one type of DNAaway from other DNA molecules

-Plasmids from bacterial chromosomal DNA(Form)

- phage DNA(Location)

-Restriction fragments, PCR products(Size)

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SDS, alkali

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alkali

neutralize

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RNA PURIFICATIONLyse & denature proteins FAST Acidic phenol

Or bind glass/silica

Small RNAs?

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A? B? C?M

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Oligo-dT beads for polyA+ mRNA

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High Salt

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Chemical Synthesisof oligonucleotides

Uses?

Block and unblock sequentiallyso that only one nucleotide addsat a time

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Phosphoramidite

5’

3’

Protected amino groups

*

*

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Couple

Growingchain

Blocked 5’-OH

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Unblock 1st “nucleotide”

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Add and activate next “nucleotide”

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Couple

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Product99%

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1% Cap

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1st nuc on bead.(blocked at 5’-OH)

unblock

couple

cap

Remove frombead

Purify

3’- 5’

Add next nuc. (blocked 5’)

De-protect

Unblock 5’-OH

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PNA

RNA harderDNA variants like

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If DNA is too large for conventional electrophoresis….

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Pulsed-field electrophoresis

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Polyacrylamide Gels can resolve smallDNAs differing inlength by onenucleotide

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Dideoxy sequencing converts sequence Information (A, C, G, T) into size differences

e.g. if a DNA has T residues at positions2, 5, 13, 16… this can be converted intoa set of DNAs of length n+ 2, 5, 13, 16..(which can be measured by denaturingpolyacrylamide gel electrophoresis)

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ddCTP

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ddATPdCTPdGTPdTTP

dATP

LABEL

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2,5,13,16

9,10,15,19

1,4,7,8,12,14,20

3,6,11,17,18

ddA

ddC

ddG

ddT

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2,5,13,16

9,10,15,19

1,4,7,8,12,14,20

3,6,11,17,18

ddA

ddC

ddG

ddT

n +

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2,5,13,16

9,10,15,19

1,4,7,8,12,14,20

3,6,11,17,18

ddA

ddC

ddG

ddT

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ddAddCddGddT

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What do you need to sequence DNA?

Where do the reagents come from?

Must the DNA be pure? How much is needed

How much good sequence can you obtain?

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Nucleic acid hybridization

Key to life and almost everyprocedure in molecular biology

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Hybridize to DNAon blot

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Recognized by specific Ab

Recognized by (strept)avidin

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RNA Probe

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NorthernRNA blot

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COLONYHYBRIDIZATION

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DNA (chromosome) in situ

FISH

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RNA in situ with non-radioactive probe