What's important in this lab?
- What is the optimal range for the spec?
- OD260=DNA absorbs light
- OD280=Protein absorbs light
- Why a Standard Curve is used?
- OD=εcd
- How to use a Spectrophotometer
- Purity Ratio= OD260/OD280
KNOW WHAT THE OPTIMAL OD (absorbance) IS FOR DNA AND PROTEIN!!!
OD260=?
OD280=?
Optimal range for the Spec: 0.1-1.0
DNA Concentration= 50ug/mL x OD units x dilution factor
***if you dilute, you MUST multiply by the df to get the ORIGINAL solution concentration.
Think of it like this..
You pour a glass of milk.
It's white, opaque, and has 100% of its Calcium. Now, say you take that milk, pour half down the sink and fill it up with water (1:2 dilution).
Half your calcium went down the drain and was replaced with water! The glass only contains 50% of the Calcium you started with!
If your mom asked you how much Calcium was in the milk in the container (the un-diluted milk), you would have to multiply concentration of Calcium in the glass you have by 2 (df) to regain the Calcium you poured down the sink and find the ORIGINAL GLASS' CALCIUM CONCENTRATION!
Get it? Got it? Good!
Why do we construct a Standard Curve???
In Parts B, we used unknown samples.
How are we supposed to figure out their concentrations?
Plot the points on the Y axis--draw a line across and down to find concentration!
REMEMBER: OD is unit-less!
Serial Dilutions are used in this lab! Check the Dilution Blog to find more info!
Using the Spec is not what we are focusing on in this lab...what is it?
Find out before you do your lab write up! Use this as a guide:
- What is the importance of OD in this lab?
- What are we determining with the OD?
- Why do we determine a purity ratio?
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