Plant Chromosome Preparation – FISH – Laser Capture Micro-dissection Workshop

Venue

Forest Tree Molecular Cytogenetics Laboratory U.S. Forest Service, Southern Research Station Southern Institute of Forest Genetics
Dept. of Ecosystem Science & Management, Texas A&M University
College Station, TX 77840

May 26 Monday

09:00 – 09:15 Welcome address by Drs. David Baltensperger, C. Dana Nelson (if available) and Thomas Byram
09:30 – 10:30 FISH presentation (Emphasis on proper plant chromosome spreading)
10:30 – 12:30 FISH Day-1 RNase Pre-treatment
Root collection – Greenhouse

12:30 – 13:30 Lunch (on your own)

13:30 – 18:00 Probe hybridization mixture preparation
Discussion: FISH with BAC, Transgene and genomic DNA probes Slide Denaturation (Chromosomal DNA)
Hybridization (DNA to DNA)
Overnight incubation
Set-up for hybridization site detection (FISH Day -2)
Root tip process for enzymatic digestion (for tomorrow)

Video Demo: Metafer (MetaSystems) – Automated detection of metaphase spreads & karyotyping

May 27 Tuesday

08:30 – 12:30 Prepare chromosome spread for FISH and Laser Capture Microdissection (LCM) of chromosome Process root tips Enzymatic digestion

12:30 – 13:30 Slide preparation

13:30 – 17:30 Lunch (on your own)
FISH discussion – Methodology & Unforeseen problems
FISH Day-2 Washing & Hybridization site(s) detection

May 28 Wednesday

08:30 – 12:00 Operation of epi-fluorescence microscope and use of MetaSystems FISH analysis software. View FISH slides, capture and process FISH images
12:00 – 13:00 Lunch (on your own)

13:00 – 17:30 View FISH slides, capture and process FISH images

May 29 Thursday

08:30 – 12:00 Principles of LCM Microscope and its operation Stain chromosome for LCM Isolation of chromosomes from slides.

12:00 – 13:00 Lunch (on your own)

13:00 – 17:30 Isolation of chromosomes from slides Amplification of captured chromosomes’ DNA

May 30 Friday

08:30 – 12:00 Clean the amplified DNA and check the concentration

12:00 – 13:00 Lunch (on your own)

13:00 – 17:30 Check the amplified DNA on an agarose gel

Particpant follow-up: Participants will be encouraged to submit a short report on the following headings within two weeks from ending the workshop:

1. Plant somatic chromosome preparation (root tip collection, pretreatment, fixation, enzymatic digestion and chromosome spread) and FISH (probe DNA, probe labeling and detection, RNase-A treatment, hybridization mixture, denaturation, hybridization and detection, capturing and pressing FISH image

2. LCM, chromosome capture and DNA amplification
The report should include at least one FISH image.
For additional details please contact the workshop organizer, e-mail: nfaridi@tamu.edu; telephone: 979-862-3908 (office), 979-575-3257 (cell).

Contact Person
Dr. Nurul Islam-Faridi
Research Plant Molecular Cytogeneticist & PI
Southern Institute of Forest Genetics 979-862-3908 (work)
979-575-3257 (cell)
Email: nfaridi@tamu.edu