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Cacao BAC Library Construction and Physical Mapping. Tissue Harvesting. 50 grams Young, expanding leaves were harvested after 12h of dark period. Nuclei Isolation. Lambda HMW DNA ladder. Chromosomal Band (Large in-tact HMW DNA). >500kb. 50 kb. Serial Digest with Hin dIII. - PowerPoint PPT Presentation
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Cacao BAC Library Constructionand
Physical Mapping
Tissue Harvesting
50 grams Young, expanding leaves were harvested after 12h of dark period
Nuclei Isolation
Chromosomal Band (Large in-tact HMW DNA)
Lambda HMW DNA ladder
50 kb
>500kb
Serial Digest with HindIIIPlugs were subject to a serial partial digestion using HindIII for 20 minutes varying the amount of enzyme. The ideal amount produces a smear from 100kb to 350kb. 1.5U HindIII for 20’ was chosen for this library.
0 2 4 6 10 100
350kb
100kb
First Size SelectionThe gel section removed was not subject to EtBr staining and UV exposure. The DNA in the size range from 150kb to 350kb was cut into 3 sections and loaded into the second size selection gel.
350kb
100kb
Second Size Selection
The center part of the gel consists of Low Melting Point Agarose. The DNA migrates into the LMP removing small, trapped fragments and will be eluted for ligation and cloning.
Recombinant ColoniesBlue/White Screening: White colonies contain BAC vector + insert, Blue colonies vector with no insert
Automated Colony Arraying: Genetix Q-Bot
BAC Characterization Gel 1
Insert
Vector
96 Random BAC clones were digested with a rare cutter and resolved by PFGE for insert size determination
BAC Characterization Gel 2
Insert Size Distribution
Insets range from 60kb-450kbAverage insert size is 140kb0% Empty Vectors
Next Steps
• Decondense 384-well plates to 96-well format• Test different restriction enzymes to determine
which combination gives the most bands per clone
• Optimize protocol for each BAC library• Begin High-Throughput Fingerprinting
Construction of Physical Map of Cacao
High-Information Content Fingerprinting (HICF) Technique:
• Restriction Digestion• SNaPshot Labeling• Capillary Electrophoresis• Fragment Analysis and Assembly
BAC library
DNA extraction
DNA digestion
DNA Labeling
Fragment analysis
FPC Capillary Electrophores
isLabeled DNA
samples run on 3730 DNA
analyzer
DNA extracted from BAC library using a 96 well alkaline-lysis
miniprep
5-enzyme restriction digest of BAC DNA
Digested DNA is labeled with 4 fluorescent dyes
(FingerPrinted Contigs)software
OVERVIEW
BAC Fingerprinting: Restriction Digest and SNaPshot Labeling
HindIII - Green
XbaI - Yellow
BamHI - Blue
XhoI - Red
HaeIII – blunt ends
AATTCGA
GATCCGG
TCGAGTC
TCAGATC
These enzymes may change once the protocol is optimized; but the overhangs would be the same.
Raw data from capillary electrophoresis processed by GeneMapper.
•High-throughput and automated•High level of accuracy
ABI3730 DNA Analyzer
Samples plot data is exported to be further analyzed in FPMiner.
Batch data analysis in FPMinerFragments are assigned a size and converted to a digitized fingerprint for contig assembly
Example of digitized fingerprint data in FPC. This
data is the contig (clones assembled because of
similar banding patterns) of one of the two control
clones we use in each plate.
Milestones and Timeline• BAC Library Construction– 1st BAC library (HindIII)— Completed– 2nd BAC library (EcoRI) — Completed– 3rd BAC library — Completed
• Preliminary data collection – In progress• Begin fingerprinting of HindIII BAC – September 8, 2008– Total number of clones to fp: 36,864– CUGI will process 4,608 clones per week
• Wetlab portion of the Physical Map of the HindIII BAC library: Estimated completion – December 1, 2008
• Data analysis portion of the Physical Map available WEBFPC : Estimated completion – January 1, 2009