biol2 lecture 1 v1
TRANSCRIPT
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Biol 2 – Lecture 1
1. Intro to Molecular Biology
2. DNA Quantification
3. Standard Curves
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What is Molecular Biology?
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Why are we studying Molecular Biology?
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What is Biol2?
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What Biol2 isn’t?
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Course Outline
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Nucleic Acid Quantification
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Nucleic Acid Quantification
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Nucleic Acid Quantification
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Nucleic Acid Quantification
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Nucleic Acid QuantificationNAMEDATE
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Nucleic Acid Quantification
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Nucleic Acid Quantification
At 260 nm,
50 µg/ml dsDNA = 1 absorbance unit
Therefore,
[dsDNA] = A260 x dilution-factor x 50 µg/ml1 abs unit
Notation Note: OD260 = A260
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What about ssDNA or RNA?
At 260 nm,
40 µg/ml RNA = 1 absorbance unit
Therefore,
[RNA] = A260 x dilution-factor x 40 µg/ml1 abs unit
33 µg/ml ssDNA = 1 absorbance unit
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Common contaminants
Phenol: λmax = 270 nm (with lots of overlap into 260 nm)
Protein: λ local max = 280 nm (mainly from Tyr)
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DNA Purity
A260
A280
λmax for DNA
λlocal max for protein
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DNA PurityFor DNA, A260 / A280
between 1.6 – 2.0 acceptable purity level
less than 1.6 probably contaminated
with Protein
greater than 2.0 probably contaminated with
Phenol or RNA
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Try it!A260 of a dsDNA sample > 1, so you decide to
make a dilution in order to lower the absorbance.
By taking 2 µl of your sample and adding it to 998
µl of dH2O, this diluted sample has an A260 of 0.138.
Q: What concentration of DNA is present in the original sample?
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[dsDNA] = A260 x dilution-factor x 50 µg/ml
1 abs unit[dsDNA] = 0.138 abs units x 1000 µl x 50 µg/ml
1 abs unit
2 µl
[dsDNA] = 0.138 x 500 x 50 µg/ml
[dsDNA] = 3450 µg/ml (or 3.45 µg/µl)
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Try it - More!The same dilution of DNA has an A280 of 0.091. Q: Is your DNA pure or not?
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A260
A280
= 0.138 = 1.51 0.091
Not quite pure enough (ratio < 1.6), probably contaminated with protein.
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Eric’s quote of the week!
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“Always read thelabel on the stock
bottle to make sureyou have theright reagent”