Human Molecular Biology Laboratory Manual

by Surzycki, Stefan

• ISBN13: 9780632046768
• ISBN10: 0632046767
• Edition: 1st
• Format: Paperback
• Copyright: 2003-01-27
• Publisher: Wiley-Blackwell

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