Molecular Biology Techniques

Please direct all comments, questions, and suggestions to biobzc@hofstra.edu

MOLECULAR BIOLOGY TECHNIQUES

Schedules of Experiments

Introduction Laboratory 1

Characterization of the Mutants

Behavioral Testing	Laboratory 1
Creation of P-element Lines and Jump-Start Mutagenesis	Laboratory 1 Assignment
Genetic Mapping of the Mutants	Laboratory 2 Assignment
Gene Expression Pattern: lacZ	Laboratory 2 and 3

Plasmid Rescue

Preparation of chromosomal DNA	Laboratory 3
Agarose gel of chromosomal DNA	Laboratory 4
Digestion of chromosomal DNA	Laboratory 4
Preparation of Competent cells	Laboratory 4
Ligation	Laboratory 4 Assignment
Transformation	Laboratory 5
Pick colonies and Start Liquid Culture	Laboratory 5 Assignment
Isolation of Plasmid DNA and test gel	Laboratory 6
Single and double digestion of plasmid DNA	Laboratory 6
Agarose gel of digested DNA	Laboratory 7
Analysis of sequencing data, database searches	Laboratory 7
Preparation of PRF of "unknown" for sequencing	Laboratory 8
Preparation of a frozen stock of new clones	Laboratory 8

Genomic Southern Analysis

Preparation of chromosomal DNA	Laboratory 3
Digestion of chromosomal DNA	Laboratory 8
Gel of digested chromosomal DNA	Laboratory 9
Genomic Southern Blot	Laboratory 9
UV crosslink DNA to membrane	Laboratory 9 Assignment
Preparation of probe	Laboratory 12
Labeling of Probe	Laboratory 13
Prehybridization and hybridization of blot	Laboratory 13
Blot washes and Detection of Non-radioactive label	Laboratory 14
Analysis of results	Laboratory 15

Northern Analysis

Preparation of Total RNA	Laboratory 7 and 8
Preparation of Poly A+ RNA	Laboratory 9
Northern Gel and Blot	Laboratory 10
UV crosslink RNA to membrane	Laboratory 10 Assignment
Preparation of probe	Laboratory 12
Labeling of Probe	Laboratory 13
Prehybridization and hybridization of blot	Laboratory 13
Blot washes and Detection of Non-radioactive label	Laboratory 14
Analysis of results	Laboratory 15

PCR Analysis of Splicing Variants

Preparation and Labeling of DNA probe

Digestion of plasmid containing common gene coding sequence and agarose gel of digested DNA	*
Isolate PRF clone from gel (GeneClean®)	Laboratory 12
Check purity of GeneClean® product by test gel	Laboratory 12
Label GeneClean® product	Laboratory 13

cDNA Library Screen

Plate Library	Laboratory 11
Phage lifts and filter preparation	Laboratory 12
Preparation of probe	Laboratory 12
Labeling of Probe	Laboratory 13
Prehybridization and hybridization of filters	Laboratory 13
Filter washes and Detection of Non-radioactive label	Laboratory 14
Analysis of results	Laboratory 15

in vivo excision of cDNA

Pick positive clones and elute	Laboratory 15
Grow host bacteria	Laboratory 15 Assignment
In vivo excision and plating	Laboratory 16
Plasmid preps of cDNA and test gel	Laboratory 17
Digestion of cDNA clones	Laboratory 17 Assignment
Test gel of digested cDNA clones	Laboratory 18

Subcloning of cDNA

Digestion of cDNA and vector for subcloning	Laboratory 18
Precipitation and Ligation reaction for subcloning	Laboratory 18 Assignment
Transformation	Laboratory 19
Plasmid DNA preparationss of subcloned cDNA	Laboratory 20
Precipitate and run test gel	Laboratory 20
Digest subcloned cDNAs	Laboratory 20 Assignment
Gel of digested subclones	Laboratory 21
Overnight culture of clone with the insert in the correct direction for expression	Laboratory 21

Muscle Protein Preparation

Dissection and glycerination of flight muscle	Laboratory 21
Muscle protein preparation	Laboratory 23
Polyacrylamide gel electrophoresis	Laboratory 23

Expression of Cloned Gene

Start large liquid culture for protein expression	Pre-Laboratory 22 Assignment
Induce expression of protein	Laboratory 22
Purification of protein on Probond® resin	Laboratory 22
SDS-PAGE gels (pre-cast)	Laboratory 23
Stain gel with Coomassie blue	Laboratory 23

* Prepared for students by technical assistant prior to laboratory when needed