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Hybridization
Community
PCR extraction
(Quantitative)
Quantification / Presence
DNA
Cloning ARDRA
Probes Sequences
5'
3'
At high temperatures, primers/probes bind to
complementary sequences (accurate)
3'
5'
At even higher temperatures heat energy is
greater than H-bounding energy and there will be
dissociation of the probe/primer template
3' 5'
Melting Curves
Percent hybridization
Perfect match
of probe/primer
Mismatched probe
T
Tm = melting temperature (point at which 50% of
probes bind to template)
PCR
Primer 1 Primer 2
Genomic DNA
gene of interest
5' 3'
dNTPs 5'
3'
DNA polymerase
}
o
Each cycle 94 C
o
72 C
Denaturation Extension o
Temp C
o o
94 C 72 C
Annealing
o
30-60 C
30-60 oC
(adjust temperature to balance between
specificity and amplification) Time
3' 5' ii
i
5' 3'
organism 1 AATT CCGG
Gene
organism 2 TCGA CCGG
3' 5'
End up with
gene of interest
Cloning
Insert into 1 2 3
plasmid Plasmids have
1 2 selective marker, like
resistance
3 to antibiotics
1 2
1 2 3
3
Pick
colonies 3
3
Extract
Multiple S
3 Grow in plasmid
liquid medium
Most abundant organisms have eluded cultivation. We only know of their existence
through cloning.