Download Southern Transfer

Agarose Gel Electrophoresis
and Southern Transfer
Broad and Long Term Objective
To determine the copy number of Myb
transcription factor genes in the genome of
the model plant Arabidopsis thaliana
Research Plan
Isolate Genomic DNA
Southern Blot
Digest Genomic DNA with Various Restriction Enzymes
Agarose Gel Electrophoresis and Southern Transfer
Make Non-Radioactive Myb Probe
Hyribidize Probe to Southern Blot
Washes and Colorimetric Detection
Data Analysis
Today’s Laboratory Objectives
1.
To become familiar with a Southern
Hybridization/Analysis
- mechanics of setting up a Southern
- what kinds of information can be gleaned
2.
To be able to evaluate a restriction digest
To distinguish between a partial and complete
digest of genomic DNA using agarose gel
electrophoresis
Theoretical Basis of Southern
Analysis
Definition
Southern analysis is the transfer of denatured DNA from an agarose gel
to a nylon or nitrocellulose membrane. This membrane is then probed
with a complementary DNA or RNA fragment that has been radioactively
or non-radioactively labeled.
Plan
1. agarose gel electrophoresis
2. membrane transfer (capillary
transfer)
3. detection (colorimetric)
Loading the Agarose Gel
Lane 1=
Lane 2=
Lane 3=
Lane 4=
Lane 5=
Lane 6=
Lane 7=
Lane 8=
Lambda Hind III Marker (negative control)
Genomic DNA/Bam HI
Genomic DNA/Eco RI
Genomic DNA/Hind III
Genomic DNA/Pst I
Genomic DNA/Eco RI + Pst I
Genomic DNA/Bam HI + Hind III
Myb61 cDNA clone (positive control)
Electrophoresis of genomic DNA
Odd numbered lanes contain undigested genomic DNA
Even numbered lanes contain digested genomic DNA
Your gel: partial or complete digest of genomic DNA?
*
Separation of DNA fragments
and preparation for capillary transfer
1. DNA fragments separated via
agarose gel electrophoresis
2. Depurinate DNA - remove adenine
and guanine residues with HCl
3. Denature DNA - separate the DNA
strands with NaOH
4. DNA neutralized w/ tris buffer
DNA Transfer Accomplished via
Capillary Action
 DNA transfer setup shown above
 DNA transfer is complete after 12-16 hours
 Electroblotting and vacuum blotting are alternative, more
rapid DNA transfer techniques
Fixation of DNA to the membrane
• After capillary transfer, single stranded DNA is loosely bound to the
nylon/nitrocellulose membrane by hydrophobic interactions between
nonpolar regions of the nylon and the exposed bases
• Hydrophobic interactions can be strengthened by removing water from the
membrane (baking or microwaving the membrane)
• DNA can be covalently linked to nylon membranes by UV crosslinking (bases
covalently bind to nylon amino groups)
Next Week
PCR will be employed to create a
non-radioactively labeled Myb61
probe
Probes will be hybridized to genomic
DNA on the nylon membrane to
determine which restriction
fragment(s) may harbor the Myb
genes