Biol 2 – Lecture 1

1. Intro to Molecular Biology

2. DNA Quantification

3. Standard Curves

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What is Molecular Biology?

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

Nucleic Acid Quantification

Nucleic Acid Quantification

Nucleic Acid Quantification

Nucleic Acid Quantification

Nucleic Acid QuantificationNAMEDATE

Nucleic Acid Quantification

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

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

Common contaminants

Phenol: λmax = 270 nm (with lots of overlap into 260 nm)

Protein: λ local max = 280 nm (mainly from Tyr)

DNA Purity

A260

A280

λmax for DNA

λlocal max for protein

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

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?

[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)

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.

Eric’s quote of the week!

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“Always read thelabel on the stock

bottle to make sureyou have theright reagent”