today do you have pcr amplicons? run gel background dna and our pcrs interpretation of pcr results...
TRANSCRIPT
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 Restriction enzymes Gabriela Perales28-Sep Southern blotting Carlos Garcia G2-Oct Cloning Timothy McBride G
6-Oct The first sequenced gene Conrad Greaves
13-Oct (q)PCR, specificity and sensitivity
Krystal Charly
16-Oct ESTs Ian Keller
20-Oct BLAST and database searches Ryan Heimroth G
23-Oct Microarrays Bianca Myers26-Oct Forensics Jennifer Gutierrez
30-OctGenome sequencing , the
$1000 genomeAyesha Arefin G
2-Nov Next generation sequencing Leslie Janet Lopez G
6-Nov Bioinformatics Amalia Parra9-Nov Epigenetics Clyde Moya
13-Nov non-coding RNA Helen Nordquist13-Nov C-value paradox Kelsey Cook G
20-Nov Phylogenetic genomics Jennifer Cooksey
23-NovGenes associated with Type 1
diabetesKatie Kesler
http://sev.lternet.edu/about
FIELDTRIP to Sevilleta LTER, Sample collection:
Sunday 13 September
(Sunday 20 September)
PARASITES AND SNAILPARASITES AND SNAIL BIOLOGY BIOLOGY
“identity, possibilities”phylogenetics
“intentions”transcriptomics
PCRrDNA/mito
BioanalyzerDNA-free,
direct sequencing
gel electrophoresisnanodrop spec
Sequence ID (BLAST)editing
Phylogenetics
electrophoresisRT-PCR
gel
CTAB/DNAzol
Trizol
TA cloning, B/W screening
M13 sequencing
Primer design, walking
Qiagen plasmid extraction Restriction digests
DNA
RNA
GenBank submission
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 MW
uneven groupPCR reactions
1-4
even groupPCR reactions
1-4
Make 1% agarose gels,1 gel/2 groups, (i.e. 1+2; 3; 5+6; 7+8; 9+10)
Use 12 well combsAnalyze 10 microliter of
each of your 4 PCR reactions.
Use layer buffer with GelRed (how much?)
5l marker/lane
Uracil lacks this group
Difference between hydrogen bonding (weak) of base pairs and covalent bonds (strong) of backbone extremely important
Biologicallyinformation is “stored” AND
can be accessed and transferred
In laboratoryds DNA can be denatured and reannealedwithout losing information
Fig. 6-15
DNA synthesis is 5’ to 3’ andsemiconservative, DNA polymerase has a proof readingcapability.
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
RNARibonucleic acidAUCGsingle stranded (5’-3’) many copies/cellvariable populationbreakdown (hydrolysis)shortmessages, regulators, transporters
DNA Deoxyribonucleic acid ATCGdouble stranded (5’-3’)one copy/cellconstant stablelongprotein encoding, introns, regulatory sequences intergenic junk(?)
NOTESProtocol gel-electrophoresis?
Can you independently repeat the PCR?
Do you know what to expect?
NOTESProtocol 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 sequencesor 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