Purifying DNA

1
Purifying DNA Basic Principles
2
Cells
Extract
Bacterial Cells Or tissue culture cells Or
blood Or flies..
HOW?
Pure DNA
3
DNA,RNA Solution
Denatured Protein
Phenol
Cell Extract
Shake
Spin
4
( salt)
SPIN (fast)
5
BIND SALT
ELUTE - NO SALT
6
EXTRACT
WATER
SILICA PARTICLES
DNA
7
pH SALT
8
Purifying one type of DNA away from other DNA
molecules

• Plasmids from bacterial chromosomal DNA
• phage DNA

9
SDS, alkali
10
(No Transcript)
11
alkali
neutralize
12
Plasmid DNA
13
RNA PURIFICATION
Lyse denature proteins FAST
14
(No Transcript)
15
(No Transcript)
16
(No Transcript)
17
A?
B?
C?
M
18
Enrichment of polyA RNA from total RNA
19
Oligo-dT beads for polyA mRNA
20
High Salt
21
(No Transcript)
22
Chemical Synthesis of oligonucleotides
Uses?
Block and unblock sequentially so that only one
nucleotide adds at a time
23
Couple
24
1st nuc on bead via 3-OH. (blocked at 5-OH)
3- 5
unblock
Add next nuc. (blocked 5)
Remove from bead
couple
De-protect
Unblock 5-OH
cap
Purify
25
Unblock 1st nucleotide
26
Phosphoramidite
Protected amino groups
5

3

27
Activate next nucleotide
28
Couple
29
Product 99
30
Cap
1
31
(No Transcript)
32
1st nuc on bead. (blocked at 5-OH)
3- 5
unblock
Add next nuc. (blocked 5)
Remove from bead
couple
De-protect
Unblock 5-OH
cap
Purify
33
PNA
34
(No Transcript)
35
(No Transcript)
36
(No Transcript)
37
(No Transcript)
38
If DNA is too large for conventional
electrophoresis.
39
Pulsed-field electrophoresis
40
(No Transcript)
41
(No Transcript)
42
Polyacrylamide Gels can resolve small DNAs
differing in length by one nucleotide
43
Dideoxy sequencing converts sequence Information
(A, C, G, T) into size differences
e.g. if a DNA has T residues at positions 2, 5,
13, 16 this can be converted into a set of DNAs
of length n 2, 5, 13, 16.. (which can be
measured by denaturing polyacrylamide gel
electrophoresis
44
(No Transcript)
45
ddCTP
46
dATP
ddATP
dCTP
dGTP
dTTP
LABEL
47
2,5,13,16
ddA
ddC
9,10,15,19
ddG
1,4,7,8,12,14,20
ddT
3,6,11,17,18
48
(No Transcript)
49
n
2,5,13,16
ddA
9,10,15,19
ddC
1,4,7,8,12,14,20
ddG
ddT
3,6,11,17,18
50
(No Transcript)
51
2,5,13,16
ddA
ddC
9,10,15,19
ddG
1,4,7,8,12,14,20
ddT
3,6,11,17,18
52
ddA
ddC
ddG
ddT
53
(No Transcript)
54
(No Transcript)
55
Cycle Sequencing differentially labeled
dideoxynucleotides
56
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?
57
Nucleic acid hybridization
Key to life and almost every procedure in
molecular biology
58
(No Transcript)
59
(No Transcript)
60
(No Transcript)
61
(No Transcript)
62
(No Transcript)
63
(No Transcript)
64
(No Transcript)
65
(No Transcript)
66
(No Transcript)
67
(No Transcript)
68
RNA Probe
69
(No Transcript)
70
Northern RNA blot
71
COLONY HYBRIDIZATION
72
DNA (chromosome) in situ
FISH
73
(No Transcript)
74
(No Transcript)
75
(No Transcript)
76
RNA in situ with radioactive probe
77
RNA in situ with non-radioactive probe