METHODS 2290F - MOLEC. BIO ROTATION

what's the formula we use to calculate cell viability?

cell viability = 1 - (# of blue cells)/(total # of cells)

- to turn this into a %, we x100

- since we're calculating a ratio, the units don't matter. it can be in cells/ml or cells/0.003ul. as long as the units are same for numerator and denom.

what's the formula we use to calculate the cell count of cells we have in our hemocytometer?

- count the # of live cells in each of the 10 squares

- average the 10 numbers

- multiply by 500

the final number is in cells/mL

what's the formula we use to calculate insert:vector ratios?

Insert mass (ng) = [(plasmid mass, ng)x(insert size, Kb)÷(plasmid size, Kb)] x (insert:vector ratio)

- Remember that Inserts Mass stands alone on the left

Insert = I

Alone = ALONE :(

what's the formula we use to calculate transformation efficiency(TE)?

TE = (# of colonies on "insert Plate") ÷ [(# of colonies on diluted TE plate)x(1/dilution factor)]

You use two plates:

1. Insert plate (NOT diluted)

- Count ALL colonies (Include blue + white)

- we only care if it transformed, we don't care if it has an insert or not (the blue colonies have no insert)

2. Diluted TE plate

- there's NO antibiotic

what's the formula we use to calculate ligation efficiency?

LE = (# white colonies on "Insert Plate") ÷ (# blue + white colonies on "Insert Plate")

- we use the insert plate only for the data in this calculation

- the insert plate is the only plate that has ligation on it

- so basically LE = (ligated e coli)/(total e coli)

How do we calculate % transmittance?

Beer-Lambert Law:

A = log₁₀(1 / %T)

- let %T be percent transmittance

- Let A be absorbance

A ∝ concentration

- The spec. 20 converts the detector signal into these values automatically.

- % T = % of light that leaves our sample

Low %T (percent transmittance) means...?

Low %T (percent transmittance) means very little light is passing through the sample.

- If less light gets through, the sample must be absorbing more, which corresponds to a higher concentration (Beer-Lambert law).

- Low %T → High absorbance → High concentration.

what instrument measures the % transmittance?

Spectrophotometer

- Any instrument that measures how light interacts with a sample

- Calculates %T and/or A

Microplate reader (a type of spectrophotometer)

- Measures % transmittance through wells in a plate

- Converts %T → absorbance

- Optimized for many samples at once

what does a standard curve plot? (what's on each axis)

The standard curve is absorbance (y) vs concentration (x).