Molecular Biology Protocols

Molecular Biology Protocols: Laboratory protocols, methods, procedures, strategies, and techniques in molecular biology, cell biology, and biochemistry. A comprehensive collection of molecular biology protocols, molecular biology methods, molecular biology techniques, molecular biology procedures, molecular biology strategies, general laboratory protocols, laboratory techniques, laboratory methods, and procedures.

Quantitative Real-time PCR in Applied Microbiology

Edited by: Martin Filion
Aimed specifically at microbiologists, this volume describes and explains the most important aspects of current real-time quantitative PCR (qPCR) strategies, instrumentation and software.

"useful book ... filled with valuable information" (Doodys) read more ...

Current Issues in Molecular Biology
Mark Strom's Molecular Biology Protocols
Jim Brown's Molecular Biology Materials and Methods
Bruce A. Roe's Molecular Biology Methods
David Bowtell's Biochemistry, Molecular Biology and Cell Biology Protocols
Brendan Babb's Collection of Protocols and Recipes
Molecular Biology Books
Other Useful Sites
References
Recommended Reading

SAGE (Serial Analysis of Gene Expression)

SAGE is a technique that allows rapid, detailed analysis of thousands of transcripts in a cell. The basic concept of SAGE rests on two principles: firstly, a small sequence of nucleotides from the transcript, called a 'tag', can effectively identify the original transcript from whence it came, and secondly, that linking these tags allows for rapid sequencing analysis of multiple transcripts. Imagine having thousands of transcripts to sequence - each sequencing event would take a certain amount of time to complete, and several thousand of these events would be necessary to identify each individual transcript. By linking the tags together, only one sequencing event is required to sequence every transcript within the cell, making the task of DNA expression profiling a much less daunting one.

Mark Strom's Molecular Biology Protocols

Mark Strom at the Microbial Pathogenesis/Utilization Research Division, Northwest Fisheries Science Center (NMFS/NOAA) maintains this excellent collection of molecular biology protocols. He also maintains a useful Molecular Biology Techniques Forum where you can submit tips, techniques, questions and comments. The methods are nicely organised into sections, including a section on computer analysis of data. Contents include:

Jim Brown's Molecular Biology Materials and Methods

Common molecular biology laboratory protocols.
This site currently contains protocols on the following topics:

ExoIII Sequencing deletions
Sequencing double-stranded DNA
Colony lifts
CsCl/Et Br plasmid isolation procedure
Isolation of total RNA using CsCl/EtBr gradients
DMS-treating DNA
DNase I footprinting
dscDNA synthesis
End-labeling DNA fragments
End-labeling RNA
Ethidium bromide spot test for DNA or RNA concentration estimation
Eubacterial RNA isolation
Exonuclease III footprinting
The figure-eight test
Giant plasmid mini-prep
Genomic DNA isolation
Glyoxal gels
Hybridization procedure #2
Preparing 5' end-labeled alkaline hydrolyzed RNA
5' End-labeling Alkaline Hydrolyzed rRNA
rRNA sequencing using reverse transcriptase
Isolating m13 clones and preparing infected cultures
Molecular Cloning Manual (Maniatis)
Culturing Methanogens
Fermenting methanogens
Micro-protein assay
Precipitation of high MW RNAs by NaCl
Nick translation
Northern gels
Nitrocellulose filter hybridization
Oligo(dt)-cellulose chromatography
32P-pCp 3' End-labeling RNA
Poly(A) assay using 3H-poly(U)
Poly(U)-sepharose chromatography
P-RNA purification
Sequencing gels
Single step prokaryotic RNA isolation
Southern blot protocol #2
Preparing spot blots
Sephadex G-50 spin column
ssDNA from pTZ/pBLUESCRIPT
Standard ligation
T7 run-off transcription
TAB restriction buffer
Transformation of CaCl2 treated E. coli cells with plasmid dna
Fixing DNA or RNA to membrane filter with UV
Saccharromyes cerevisiae total rna isolation
Mini cleared lysate protocol
Oligo(dT) cellulose batch chromatography
32P-cDNA synthesis

Bruce A. Roe's Molecular Biology Methods Click here

Bruce A. Roe, Department of Chemistry and Biochemistry, University of Oklahoma, Norman, Oklahoma 73019, broe@uoknor.edu, has compiled this really useful manual of many of the everyday methods used in the average molecular biology laboratory, with emphasis on the techniques for large scale DNA sequencing protocols and DNA sequencing automation techniques. The manual has been written in a protocol format, with little theoretical discussion. For theory and additional information, users of this manual are referred back to the original literature.

Table of Contents

I. General methods
A. Phenol extraction of DNA samples
B. Concentration of DNA by ethanol precipitation
C. Restriction digestion
D. Agarose gel electrophoresis
E. Elution of DNA fragments from agarose
F. Kinase end-labeling of DNA
G. Bacterial cell maintenance
H. Fragment purification on Sephacryl S-500 spin columns
II. Random subclone generation
A. Sonication
B. Nebulization
C. Random fragment end-repair, size selection, and phosphorylation
D. DNA ligation
E. Competent cell preparation
F. Bacterial cell transformation
G. Microcentrifuge tube transformation
III. Methods for DNA isolation
A. Large scale double-stranded DNA isolation
B. Midiprep double-stranded DNA isolation
C. Miniprep double-stranded DNA isolation
D. Large scale M13RF isolation
E. Single-stranded M13 DNA isolation using phenol
F. Biomek-automated modified-Eperon isolation procedure for single-stranded M13DNA
G. 96 well double-stranded template isolation
H. Genomic DNA isolation from blood
IV. Methods for DNA sequencing
A. Bst-catalyzed radiolabeled DNA sequencing
B. Radiolabeled sequencing gel preparation, loading, and electrophoresis
C. Taq-polymerase catalyzed cycle sequencing using fluorescent-labeled dye primers
D. Taq-polymerase catalyzed cycle sequencing using fluorescent-labeled dye terminator reactions
E. Sequenase[TM] catalyzed sequencing with dye-labeled terminators
F. Fluorescent-labeled sequencing gel preparation, pre-electrophoresis, sample loading, electrophoresis, data collection, and analysis on the ABI 373A DNA sequencer
G. Double-stranded sequencing of cDNA clones containing long poly(A) tails using anchored poly(dT) primers
H. cDNA sequencing based on PCR and random shotgun cloning >V. Additional methods
A. Polymerase Chain Reaction (PCR)
B. Purification of PCR fragments for cloning
C. Preparation of SmaI-linearized, dephosphorylated double-stranded M13 replicative form cloning vector
D. Synthesis and purification of oligonucleotides
E. Rapid hybridization of complementary M13 inserts
APPENDIX
Solutions
Primers
Taq Cycle Sequencing Reagent Preparation
Oligonucleotide universal primers used for DNA sequencing
Listing of M13 (pUC) cloning sites
Commonly used restriction enzymes and assay buffers
Bacterial Transformation and Transfection
Units and formulas
DNA mobility in gels
Codon chart and amino acid symbols
Biomek configuration for single stranded DNA isolation
Consensus sequences in nucleic acids
References

David Bowtell's Biochemistry, Molecular Biology and Cell Biology Protocols

Another great collection of protocols on the web. This one is maintained by David Bowtell and contains protocols in Biochemistry, Molecular Biology and Cell Biology. These include:

Isolation of DNA fragments using glass milk (GENE-CLEAN)
SOUTHERN BLOT
SOUTHERN ANALYSIS OF MOUSE TOE/TAIL DNA
Northern transfer protocol
Hybridizing Northern blots with riboprobes
cDNA + GENOMIC DNA LIBRARY SCREENING
Isolation of Mouse genomic DNA
RNA ISOLATION USING GUHCL
POLY A+ RNA PREPARATION using proteinase K and SDS
SELECTION OF POLYADENYLATED RNA FROM TOTAL RNA
Mini plasmid preps
Large Scale Plasmid Preps
Preparing and USING bacterial cells for electroporation
In situ hybridisation to RNA in tissue sections
PRIMER LABELLING FOR PRIMER EXTENSION ASSAYand PRIMER EXTENSION
DNA-se I FOOTPRINTING
Double stranded DNA sequencing (Dideoxy)
Buffer gradient SEQUENCING GELS
Nested Deletions using exonuclease-III and mung bean nuclease
SITE DIRECTED MUTAGENESIS USING THE UNG - DUT SYSTEM.
In vitro mutagenesis using Altered Sites
Labeling of oligonucleotides
RANDOM HEXAMER LABELLING OF CDNA PROBES
RNAse protection assay
Isolation of phage DNA
PHAGE PURIFICATION WITH DE52
BUTANOL PRECIPITATION OF DEPROTECTED OLIGONUCLEOTIDES
OLIGO PURIFICATION on acrylamide gels
POLYMERASE CHAIN REACTION
Rapid Amplification of mRNA ENDS by PCR (RACE)
YEAST MEDIA
TRANSFORMATION OF YEAST
Modified yeast transformation
Isolation of shuttle vectors from yeast and transformation into E. coli
Yeast lysates for westerns
Biochemical techniques:
SDS-PAGE gels
Reagents for Protein Mini Gels
Western transfer using a semidry apparatus.
Affinity purification of antibodies from crude serum
Immunoprecipitation from eukaryotic cell extracts.
Subcellular Fractionation of proteins
Serum starving Her14 cells prior to EGF stimulation
Preparation of PGEX bacterial Lysates.
Purification of GST Fused Proteins
Thrombin cleavage of GST fused protein
Biotinylation of pGEX proteins
POLYCLONAL AND MONOCLONAL ANTIBODIES
Expanding Hybridoma Clones
Cell Biology Techniques
MEDIA AND SOLUTIONS REQUIRED FOR ROUTINE ES CELL CULTURE
Routine Culturing of ES Cells
ISOLATION OF PRIMARY MOUSE EMBRYO FIBROBLASTS
MITOMYCIN C TREATMENT OF PMEFs
GAMMA IRRADIATION OF PMEF'S
PMEF FEEDER LAYER CONCENTRATION
ELECTROPORATION OF ES CELLS AND ISOLATION OF H/R CLONES
KARYOTYPING ES CELLS
VASECTOMY OF MALE MICE
Normaski Optics
MEDIA FOR EMBRYO CULTURE AND MANIPULATION
Suppliers
PREPARATION OF MICROINJECTION PIPETTES
EMBRYO MANIPULATION PIPETTES (FLUSHERS ETC)
COLLECTION OF MORULAE and Earlier
Aggregation of ES cells and eight cell stage embryos
BLASTOCYST TRANSFER
Sending sera to testing for mouse pathogens.
SUPEROVULATION OF MICE
FLUSHING OUT OF BLASTOCYSTS
Alizarin Red S (bone) and alcian blue (cartilagestaining of cleared skeletons.
Routine Splitting and freezing of cells
SOFT AGAR ASSAY FOR COLONY FORMATION
VIRUS INFECTION OF FIBROBLASTS
DNA TRANSFECTION OF EUKARYOTIC CELLS USING CALCIUM PHOSPHATE
LIPOFECTAMINE FOR TRANSFORMATION OF EUKARYOTIC CELLS
ELECTROPORATION OF COS-7 CELLS
Growth and transfection of PC12 cells.
CELL CULTURE FROM WHOLE MICE EMBRYO (DAY 10 - 11 PC)
PCR GENOTYPING FROM the EMBRYO YOKE SACK
b-gal staining of eukaryotic cells in vitro
Comments on db-cAMP and forskolin
Comments on Ro 31.8220
IMMUNOFLUORESENCE ON CHAMBER SLIDES
PHOTOMICROGRAPHY
Reagents

Brendan Babb's Collection of Protocols and Recipes

A comprehensive collection of molecular biology techniques, recipes and protocols arranged in alphabetical order. Submissions and comments are always welcome. The pages have been assembled by Brendan Babb at the department of Medical Microbiology, University of Cape Town.

Protocols:

I. Bacterial methods
DNA isolation, transformation, RNA isolation...

II. DNA related protocols
Restriction, ligation, Southern blots, ...

III. RNA related protocols
Northern blots, primer extension, ...

IV. Protein related protocols
SDS-PAGE gels, Western blots, ...

V. Polymerase Chain Reaction (PCR)
Descriptions and discussion of PCR techniques,

Recipes

A
acrylamide gel mix
Acrylamide stock
Ammonium acetate
Ammonium persulphate
Ampicillin
ATP
B
Bal31 dilution/storage buffer
Bal31 reaction buffer
Blocking buffer stock solution, Westerns
Blocking buffer, Westerns
Blot buffer, Westerns
BSA (bovine serum albumin)
C
Calcium chloride, CaCl2
Cell Suspension buffer
Chloroform-isoamyl alcohol mix (24:1)
Church hyb buffer
Church prehyb buffer CHB
CTAB/NaCl (hexadecyltrimethyl ammonium bromide)
Cysteine
D
Denaturation solution 2 (0.4M NaOH/1M NaCl)
Destain, SDS-PAGE
Dithiothreitol, DTT
DNA digestion: core reaction buffers
DNA digestion: enzyme dilution buffer
E
EDTA
Ethanol
Ethidium bromide, EtBr
ExoIII reaction buffer
F
G
Gel mixes, SDS-PAGE
Glucose
H
Hybridization solution (Zappe)
I
IPTG, Isopropyl-�-D-thio-galactopyranoside
Isopropanol, salt-saturated
J
K
Klenow buffer (10x)
Klenow mix
L
Laural sulphate, see sodium dodecyl sulphate
Ligation dilution buffer
Ligation mix
Ligation reaction buffer
Luria broth, LB
Lysing solution
M
Magnesium chloride, MgCl2
Magnesium sulphate, MgSO4
Minimal medium, E. coli
Minimal medium 5x salts, E. coli
Mungbean nuclease dilution/storage buffer
Mungbean nuclease reaction buffer
N
O
P
PB (phosphate buffer)
Phenol Chloroform mix
Phenol (buffered pH 7.6, 10mM Tris)
Potassium chloride
Potassium salts
Q
R
Resolving buffer, SDS-PAGE
RNAse mix
S
S1 mix
S1 stop mix
Salt, see sodium chloride
Sample buffer, SDS-PAGE
SDS, see sodium dodecyl sulphate
Sephadex G-50
Sma I reaction buffer
Sodium acetate, NaAc
Sodium chloride, NaCl
Sodium dodecyl sulphate, SDS
Sodium hydroxide, NaOH
Sodium perchlorate, NaClO4
Solution III, plasmid preps
Solution II, plasmid preps
Solution I, plasmid preps
SSC (salt sodium citrate) 20x
ST1 Southern transfer solution 1
ST2 Southern transfer solution 2
ST3 Southern transfer solution 3
Stacking buffer, SDS-PAGE
Stain, SDS-PAGE
STE
T
TA buffer
TAE buffer,Tris-acetate-EDTA
Tank buffer, SDS-PAGE
Taurine buffer (TTE)
TB buffer
TBE buffer
TE buffer, Tris-EDTA buffer
TEMED
Terrific broth
Tracking dye (IV)
Tracking dye (Coyne)
Tris buffer
Tris-Base
tRNA
TSB, TSBG
U
V
W
Wash buffer A (WBA)
Wash buffer B (WBB)
Wash buffer C (WBC)
X
X-gal, 5-bromo-4-chloro-3-indolyl-�-D-galactoside
Y
YT 2x, yeast tryptone medium
Z
Zinc acetate
Zinc sulphate

Nest Group Protocols Click here

A collection of products, applications, and protocols from the Nest Group

Kits for Nucleic Acid Extraction
Nucleobond AX* properties and high purity applications
Nucleobond AX* - trouble shooting guide
NucleoTrap* and NucleoTraPCR* properties and applications
NucleoSpin* and NucleoSpin Extract* properties and applications
Protein & Peptide HPLC Column Applications:
HILIC and SEC Columns PolySULFOETHYL and HYDROXYETHYL Spec Sheet Polyaspartic Acid* Weak Cation Exchange Spec Sheet
PolySULFOETHYL Aspartamide* Strong Cation Exchange Spec Sheet (ALKYL) Aspartamide* HIC Spec Sheet
PolyWAX LP* Weak Anion Exchange Spec Sheet
PolyGLYCOPLEX* Complex Carbohydrates Spec Sheet
SDS Removal Guard Column Spec Sheet
PolyLC Part Numbers and Prices
Validation of Reverse Phase Chromatography separations (TFA, PO4, HOAc, HCl) Spec Sheet