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9780632046768

Human Molecular Biology Laboratory Manual

by
  • ISBN13:

    9780632046768

  • ISBN10:

    0632046767

  • Edition: 1st
  • Format: Paperback
  • Copyright: 2003-01-27
  • Publisher: Wiley-Blackwell

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Summary

Human Molecular Biology Laboratory offers a hands-on, state-of-the-art introduction to modern molecular biology techniques as applied to human genome analysis. In eight unique experiments, simple step-by-step instructions guide students through the basic principles of molecular biology and the latest laboratory techniques. This laboratory's distinctive focus on human molecular biology provides students with the opportunity to analyze and study their own genes while gaining real laboratory experience.

Author Biography

Stefan Surzycki is the author of Human Molecular Biology Laboratory Manual, published by Wiley.

Table of Contents

Preface xi
Preparation of Human Genomic DNA
1(19)
Introduction
1(1)
Background
1(2)
Cell Breakage
3(1)
Removal of Protein
3(3)
Deproteinization using organic solvents
3(3)
Deproteinization using enzymes
6(1)
Removal of RNA
6(1)
Concentrating the DNA
7(2)
Determination of the Purity and Quantity of DNA
9(2)
First Laboratory Period
11(5)
Safety precautions
11(1)
Technical tips
11(2)
Protocol
13(3)
Second Laboratory Period
16(4)
Safety precautions
16(1)
Technical tips
16(1)
Protocol
16(2)
Expected results
18(1)
References
19(1)
DNA Fingerprinting: Multi-locus Analysis
20(42)
Introduction
20(1)
Background
20(5)
First Laboratory Period
25(12)
restriction enzyme digestion
25(1)
Introduction
25(1)
Background
25(1)
Technical tips
26(1)
Protocol
27(1)
References
28(1)
agarose gel electrophoresis
29(1)
Introduction
29(1)
Background
29(5)
Safety precautions
34(1)
Protocol
34(2)
References
36(1)
Second Laboratory Period
37(6)
Southern blotting
37(1)
Introduction
37(1)
Background
37(1)
Safety precautions
38(1)
Technical tips
38(1)
Protocol
38(3)
References
41(2)
Third Laboratory Period
43(13)
preparation of probe and hybridization
43(1)
Introduction
43(1)
Background
43(7)
Technical tips
50(1)
Protocol
51(3)
References
54(2)
Fourth Laboratory Period
56(2)
Protocol
56(2)
Fifth Laboratory Period
58(4)
Protocol
58(1)
Data analysis
59(3)
DNA Fingerprinting: Single-locus Analysis
62(14)
Introduction
62(1)
Background
63(3)
First Laboratory Period
66(3)
Protocol
66(3)
Second Laboratory Period
69(7)
Protocol
69(1)
Data analysis
69(3)
Expected results
72(3)
References
75(1)
General reading
75(1)
Out of Africa: Origin of Modern Humans
76(19)
Introduction
76(1)
Background
76(9)
Origin of humans
76(4)
PCR
80(5)
First Laboratory Period
85(4)
Technical tips
85(1)
Protocol
85(4)
Second Laboratory Period
89(6)
Safety precautions
89(1)
Protocol
89(3)
Data analysis
92(1)
Expected results
93(1)
References
94(1)
DNA Sequencing
95(44)
Introduction
95(1)
Background
96(4)
DNA sequencing methods
97(1)
Sequencing strategies
98(1)
References
99(1)
First Laboratory Period
100(11)
nebulization shearing of DNA
100(1)
Introduction
100(1)
Background
100(2)
Safety precautions
102(1)
Technical tips
103(1)
Protocol
104(2)
Expected results
106(1)
repair of the ends of sheared DNA
106(1)
Introduction
106(1)
Safety precautions
106(1)
Technical tips
107(1)
Protocol
108(2)
References
110(1)
Second Laboratory Period
111(19)
ligation to sequencing vector
111(1)
Introduction
111(1)
Background
111(7)
Technical tips
118(1)
Protocol
119(2)
References
121(1)
transformation of bacteria by electroporation
121(1)
Introduction
121(1)
Background
122(2)
Technical tips
124(2)
Protocol
126(2)
Expected results
128(1)
References
129(1)
Third Laboratory Period
130(7)
preparation of plasmid for DNA sequencing
130(1)
Introduction
130(1)
Background
130(1)
Technical tips
131(1)
Protocol
132(2)
sequencing reactions for an ABI 3700 sequencer
134(1)
Introduction
134(1)
Technical tips
135(1)
Protocol
136(1)
Fourth Laboratory Period
137(2)
removing dideoxy terminators
137(1)
Protocol
137(1)
References
137(2)
Computer Analysis of Sequencing Data
139(25)
Introduction
139(1)
Background
139(19)
Databases and sequence formats
140(3)
Sequence alignments
143(4)
BLAST
147(3)
FASTA
150(1)
BLAST versus FASTA
150(1)
Single-sequence analysis
151(1)
Dot matrix analysis
152(2)
Technical tips
154(1)
Protocol
155(2)
References
157(1)
Sequence Alignment with BLAST
158(1)
Search of ``nr'' database
158(1)
Search for an Alu SINE element
159(1)
Search for expressed sequences
159(1)
Search for the Chromosome Position of the Query Sequence
159(2)
Single-sequence Analysis
161(3)
Converting file formats
161(1)
Base content analysis
162(1)
Restriction enzyme site analysis
162(1)
Dot matrix analysis
162(2)
Determination of Human Telomere Length
164(32)
Introduction
164(1)
Background
165(5)
First Laboratory Period
170(4)
isolation of genomic DNA
170(1)
Introduction
170(1)
Background
170(1)
Safety precautions
171(1)
Protocol
171(3)
Second Laboratory Period
174(6)
determination of DNA concentration and purity
174(1)
Protocol
174(1)
restriction enzyme digestion
174(1)
Introduction
174(1)
Background
175(1)
Technical tips
175(1)
Protocol
176(1)
agarose gel electrophoresis
177(1)
Introduction
177(1)
Background
177(1)
Safety precautions
177(1)
Technical tips
177(1)
Protocol
178(2)
Third Laboratory Period
180(5)
Southern transfer
180(1)
Introduction
180(1)
Background
180(1)
Safety precautions
180(1)
Technical tips
181(1)
Protocol
181(4)
Fourth Laboratory Period
185(2)
DNA hybridization
185(1)
Introduction
185(1)
Background
185(1)
Technical tips
185(1)
Protocol
186(1)
Fifth Laboratory Period
187(3)
Protocol
187(3)
Sixth Laboratory Period
190(6)
analysis of TRF length
190(1)
Introduction
190(1)
Background
190(1)
Technical tips
191(1)
Protocol
192(2)
References
194(2)
RT-PCR of Human Genes
196(19)
Introduction
196(1)
Background
196(2)
First Laboratory Period
198(11)
purification of total RNA
198(1)
Introduction
198(1)
Background
198(2)
Safety precautions
200(1)
Technical tips
201(1)
Protocol
202(1)
References
203(1)
RNA agarose gel electrophoresis
204(1)
Introduction
204(1)
Background
204(1)
Safety precautions
205(1)
Technical tips
205(1)
Protocol
205(2)
References
207(2)
Second Laboratory Period
209(6)
running an RT-PCR
209(1)
Introduction
209(1)
Background
209(3)
Safety precautions
212(1)
Technical tips
212(1)
Protocol
213(2)
Appendix 215(8)
DNA Purification
215(2)
Equipment and supplies
215(1)
Solutions to prepare
216(1)
DNA Fingerprinting: Multi-locus Analysis
217(2)
Equipment and supplies
217(1)
Solutions to prepare
218(1)
DNA Fingerprinting: Single-locus Analysis
219(1)
Equipment and supplies
219(1)
Solutions to prepare
219(1)
Out of Africa: Origin of Modern Humans
220(1)
Equipment and supplies
220(1)
Solutions to prepare
220(1)
DNA Sequencing
220(2)
Equipment and supplies
220(1)
Solutions to prepare
221(1)
Determination of Human Telomere Length
222(1)
Equipment and supplies
222(1)
RT-PCR of Human Genes
222(1)
Equipment and supplies
222(1)
Index 223

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