Download Today - Biology

Today
• HOUSEKEEPING,
background, presentations
• DNA extraction from snails
• DNA extraction from parasites (cercariae)
• Start protocols (timing!)
• Background on methods
• Complete extractions protocol
PARASITES AND SNAIL BIOLOGY
DNA
“identity, possibilities”
phylogenetics
RNA
“intentions”
transcriptomics
CTAB/DNAzol
Trizol
gel electrophoresis
nanodrop spec
Bioanalyzer
DNA-free,
PCR
rDNA/mito
TA cloning, B/W screening
electrophoresis
direct sequencing
Sequence ID (BLAST)
editing
Phylogenetics
GenBank
submission
Qiagen plasmid extraction
Restriction digests
M13 sequencing
Primer design, walking
RT-PCR
gel
Shady Lakes: SNAILS AND PARASITES
Physid
Specimens
infected snails +
released cercariae
fixed in 80% EtOH
Physella sp?
Wethington Leydeard,
2007
Lattitude 35°12'59.15"N
Longitude 106°35'54.52"W
(Google Earth)
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Things to be careful about with
DNA Extraction
Safety
Vortexing/pipetting
Too much starting material?
When is a good time to stop
Contamination
Add too much of a reagent? Don’t freak out
What should you use to reconstitute or dissolve
your DNA?
• Where is the DNA?
• Solution of DNA too concentrated/too dilute
SAMPLE ASSIGNMENTS
2(3)
6
7
SP5/PP5
SP4/PP4
SP2/PP2
10
SP2/PP2
8
SP4/PP4
1
SP1/PP1
5
SP5/PP5
9
SP1/PP1
EXTRACT DNA
HANDOUT 3
complete step 1-6 of DNA extraction
15 minute powerpoint topics
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Discovery of DNA structure
Restriction enzymes
Southern blotting
Cloning
The first sequenced gene
PCR, specificity and sensitivity
RAPDs
q-PCR
BAC libraries
ESTs
BLAST and database searches
Microarrays
Forensics
Genome sequencing , the $1000 genome
Next generation sequencing
Bioinformatics
Epigenetics
"non-coding" RNA
C-value paradox
Phylogenetic genomics
Archeological genomics
YOUR favorite gene (check with instructor)
Research your topic
(Coen provides guidance on
literature)
Prepare, present ppt
presentation on topic
(12 + 3 format)
Participate in Q and A
Start Sept 22nd
Sample Preservation
• Collect from the field, freeze at -70C
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– alternatives
Extraction buffer - good DNA, unhappy airlines
RNA later (AMBION) - good RNA (DNA)
70-100% ethanol - dehydrates DNA
Guanidinium isothiocyanate -good for RNA
Dry - varying results (good/low yield/degradation)
Formaldehyde - Bad: cross-links (formic acid,
depurinates DNA (A,G)
DNA/RNA WHERE?
RNA
DNA
(shell)
(mechanical
tissue
disruption)
cells
nucleus/mitochondria
Also in the way, lipids, mucopolysaccharides, proteins (enzymes)
Avoid all that and obtain DNA/RNA
parasites/other symbionts, animal, plant, bacterial, fungal?
DNA Extraction
CTAB-cetyltrimethylammonium bromide, cationic
detergent, forms complexes with (poly)saccharides
(and protein?) to help mucopolysaccharide removal
EDTA-ethylene di-amino tetra acetic acid,
chelates divalent metals, cofactors for nucleases
Tris/HCl pH 8.0 - maintains pH (important for DNA)
Beta mercapto ethanol - lyses cells, denatures proteins
Proteinase K- degrades proteins (at 60C!)
NaCl - helps precipitation
High temperature - inactivates enzymes
DNA isolation
• Industry provides BLACK BOX
• SDS (detergent) dissolves lipids
• Chaotropic salts denature proteins, disrupt
protein structure, cluster nucleic acids
A chaotropic agent, also known as chaotropic reagent and
chaotrope, is a substance which disrupts the three dimensional
structure in macromolecules such as proteins, DNA, or RNA and
denatures them.
Chaotropic agents interfere with stabilizing intra-molecular interactions
mediated by non-covalent forces such as hydrogen bonds, van der
Waals forces, and hydrophobic effects.
Chaotropic reagents include:
http://www.mrcgene.com/dnazoffer.htm
Urea
Guanidinium chloride
http://en.wikipedia.org/wiki/Chaotropic_agent
Organic (Chloroform) Extraction
• Mix snail extract with chloroform,
centrifuge to separate into phases.
DNA (>pH8.0)
RNA
proteins
chloroform
debris, other
Alcohol precipitation
Alcohol plus aqueous (UPPER) phase
DNA cannot retain water in the presence of
alcohol, salt and will precipitate out
How it works physico-chemically, see
http://bitesizebio.com/253/the-basics-how-ethanol-precipitation-of-dna-and-rna-works/
EtOH (100-95%) or isopropanol (100%)
Clean-up and dissolve in molecular grade H2O
DNA and RNA
• DNA from nucleus AND mitochondria (identity
and genomics)
• Organic extraction needs pH 8 or higher
• (+ RNA) RNA transcribed from genes
• RNA cytosol (intentions and regulation)
• Also at < pH 8.0
• (+ DNA) different in structure, susceptible to
break down, degradation.
NEW kits greatly facilitate working with RNA,
RNA later, RNAseZAP.
NUCLEIC ACIDS QUALITY?
• See fibers, pellets? Or not?
• Quality, Amount, Composition?
• Gelelectrophoresis
• Spectrophotometry