Today
• Do you have PCR amplicons?
• Run gel
• Background DNA and our PCRs
• Interpretation of PCR results
• What to do next?
15 minute powerpoint topics(G+)
date
topic
name
21-Sep
Discovery of DNA structure
Janette Mendoza
25-Sep
28-Sep
2-Oct
Restriction enzymes
Southern blotting
Cloning
Gabriela Perales
Carlos Garcia
Timothy McBride
6-Oct
The first sequenced gene
Conrad Greaves
13-Oct
16-Oct
(q)PCR, specificity and
sensitivity
ESTs
20-Oct
BLAST and database searches
Ryan Heimroth
23-Oct
26-Oct
Microarrays
Forensics
Bianca Myers
Jennifer Gutierrez
30-Oct
Genome sequencing , the
$1000 genome
Ayesha Arefin
G
2-Nov
Next generation sequencing
Leslie Janet Lopez
G
6-Nov
9-Nov
13-Nov
13-Nov
Bioinformatics
Epigenetics
non-coding RNA
C-value paradox
Amalia Parra
Clyde Moya
Helen Nordquist
Kelsey Cook
G
20-Nov
Phylogenetic genomics
Jennifer Cooksey
23-Nov
Genes associated with Type 1
diabetes
Katie Kesler
G
G
Krystal Charly
Ian Keller
G
http://sev.lternet.edu/about
FIELDTRIP to Sevilleta LTER, Sample collection:
Sunday 13 September
(Sunday 20 September)
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
Today
• Use your notes/handouts
• Make 1% agarose gels, 1 per 2 groups
(i.e. 1+2; 3; 5+6; 7+8; 9+10 )
• Analyze 10 microliter of each of your 4
PCR reactions. Use layer buffer with
GelRed (how much?)!
• Lecture
• Results
MW
Make 1% agarose gels,
1 gel/2 groups, (i.e. 1+2;
3; 5+6; 7+8; 9+10)
Use 12 well combs
Analyze 10 microliter of
each of your 4 PCR
reactions.
Use layer buffer with
GelRed (how much?)
5ml marker/lane
MW
uneven group
PCR reactions
1-4
even group
PCR reactions
1-4
Uracil lacks this group
Difference between hydrogen bonding (weak) of base
pairs and covalent bonds (strong) of backbone extremely
important
Biologically
information is “stored” AND
can be accessed and transferred
In laboratory
ds DNA can be denatured and reannealed
without losing information
Fig. 6-15
DNA synthesis is 5’ to 3’ and
semiconservative, DNA polymerase has a proof reading
capability.
DNA replication = DNA synthesis (template = DNA)
(transfers information from generation to generation--cell and
organism) Primer required
Transcription = RNA synthesis (template = DNA)
(transfers information from DNA to cellular metabolic machinery)
Promoter not primer required
DNA
RNA
Deoxyribonucleic acid
ATCG
double stranded (5’-3’)
one copy/cell
constant
stable
long
protein encoding, introns,
regulatory sequences
intergenic junk(?)
Ribonucleic acid
AUCG
single stranded (5’-3’)
many copies/cell
variable population
breakdown (hydrolysis)
short
messages, regulators,
transporters
NOTES
Protocol gel-electrophoresis?
Can you independently repeat the PCR?
Do you know what to expect?
NOTES
Protocol gel-electrophoresis?
YES
Can you independently repeat the PCR?
Do you know what to expect?
NO and NO
Because you were not given the needed
information for the PCR reaction,
the primer sequences
or size estimates of the anticipated amplicons
PCR details
• AmpliTaq Gold,
4 mM MgCl2
• 1 mM of each primer (50 picomoles)
• Cycling profile
–
–
–
–
10' 95C (hot start)
30x (30" 95C; 60" Tm; 60"+5" 72C)
7' 72C,
∞ 4C
PCR details
• Primers:
• 16S and CO1
– 16S Tm 55C expected size ~600bp
• 16SAr: 5'- CGC CTG TTT ATC AAA AAC AT -3’
• 16SBr: 5'- CCG GTC TGA ACT CAG ATC ACG T -3’
(Palumbi, S. R. 1996. Nucleic acids II: the polymerase chain reaction. In: Molecular Systematics
(eds. Hillis, D. M., Moritz, C. and Mable, B. K.), pp. 205–247. Sinauer & Associates Inc., Sunderland, Massachusetts.)
– CO1 Tm 48C expected size ~700bp
• LCO1490: 5'-GGT CAA CAA ATC ATA AAG ATA TTG G -3’
• HC02198: 5'-TAA ACT TCA GGG TGA CCA AAA AAT CA-3’
(Folmer O, Black M, Hoeh W, Lutz R, Vrijenhoek R. 1994 DNA primers
for amplification of mitochondrial cytochrome C oxidase subunit I from diverse metazoan invertebrates. Molecular Marine
)
Biology and Biotechnology, 3, 294–299
PCR details
• Primers:
• "parasite" rDNA 18S and 28S (Olsen et al. 2003).
– 18S Tm 50C expected size ~1800bp
• wormA: 5'- A/GCG AAT GGC TCA TTA AAT CAG -3’
• wormB: 5'- ACG GAA ACC TTG TTA CGA CT -3’
– 28S Tm 50C expected size ~1400bp
• LSU: 5'- TAG GTC GAC CCG CTG AAY TTA AGC A -3’
• 1500R: 5'- GCT ATC CTG AGG GAA ACT TCG -3’
PCR interpretation
Band?
Single/multiple?
Strong/weak?
Correct size?
Positive ID?
WHERE TO GO NOW