Lab Two: SPECTROPHOTOMETRY

Spec-tro-pho-tom-etry...sound it out.

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?

Lab One: SOLUTION MAKING!

I know what you thought when you started this lab. "Is this a joke? How dumb! I know how to dilute something, and why the hell are we making solutions the first day of lab? We are supposed to get out EARLY!!!"

Well I hope you didn't slack on those calculations because all those solutions will be used later this semester....in YOUR experiments!!!

So let's talk about dilution calculations...

Harder than it seems, right? But I'm going to break it down.

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Volume to Volume Dilutions

This type of dilutions describes the ratio of the solute to the final volume of the diluted solution.

For example, to make a 1:10 dilution of a 1M NaCl solution, you would mix one "part" of the 1M solution with nine "parts" of solvent (probably water), for a total of ten "parts." Therefore, 1:10 dilution means 1 part + 9 parts of water (or other diluent).



For example: if you needed 10 mL of the 1:10 dilution, then you would mix 1mL of the 1M NaCl with 9mL of water.

Or: if you needed 100mL of the 1:10 dilution, then you would mix 10mL of the 1M NaCl with 90mL of water.

The final concentration of NaCl in both cases is 0.1M.

C1V1=C2V2

The Big Boy of Dilutions...know this!!!

Sometimes it is necessary to use one solution to make a specific amount of a more dilute solution. To do this, you can use the formula:

V1C1 = V2C2

where:
V1 = volume of starting solution needed to make the new solution
C1 = concentration of starting solution
V2 = final volume of new solution
C2 = final concentration of new solution

For example: Make 5mL of a 0.25M solution from 2.5mL of a 1M solution.

V1C1 = V2C2
(V1)(1M) = (5mL)(0.25M)
V1 = [(5mL)(0.25M)] / (1M)
V1 = 1.25mL

So you will need to use 1.25mL of the 1M solution. Since you want the diluted solution to have a final volume of 5mL, you will need to add ( V1-V2 = 5mL - 1.25mL) 3.75mL of diluent.

Serial Dilutions

Sometimes it is necessary to dilute by a large factor so several serial dilutions are necessary.
To make a 1:10 dilution a 1:100 via serial dilution...

Take 1 part diluted to 9 parts water! This is another 1:10 dilution!

1:10 x 1:10= 1:100

Solutions made using Percentage by weight (w/v)

The number of grams in 100mL of solution is indicated by the percentage.
For example, a 1% solution has one gram of solid dissolved in 100mL of solvent. To make this type of solution properly, you should weight 1g and dissolve it in slightly less than 100mL. Once the solids have dissolved, you can bring the volume up to the final 100mL

Solutions made using Percentage by Volume (v/v)

In this case, the percentage indicates the volume of the full strength solution in 100mL of dilute solution.

To make a 60% Ethanol solution:

Step 1: Measure 60mL ethanol

Step 2: Add 40mL water to bring to 100mL

Molar Solutions

A 1 molar solution is a solution in which 1 mole of a compound is dissolved in a total volume of 1 litre.

For example:
The molecular weight of sodium chloride (NaCl) is 58.44, so one gram molecular weight (= 1 mole) is 58.44g. If you dissolve 58.44g of NaCl in a final volume of 1 litre, you have made a 1M NaCl solution.

To make a 0.1M NaCl solution, you could weigh 5.844g of NaCl and dissolve it in 1 litre of water; OR 0.5844g of NaCl in 100mL of water (see animation below); OR make a 1:10 dilution of a 1M sample.

Do it on your own:

How would you make a 500mL dilution of a 0.05M NaCl solution?

Answer will be posted when someone figures it out! Post your answers in the comment section!

Courtesy of http://www.wellesley.edu/Biology/Concepts/Html/volumetovolume.html

HOLD UP! How does this work??

So here's the deal...

Every lab will have its own blog. Questions, comments, interesting facts, and epiphanies can be posted in the "Comments" section.

How to ask a question:

• Click the "comments" link
• Type your question in "Comments" box
• Choose a username to post as (AIM screen name, google account, etc.) in the drop box.
• If you don't have one of these, create a google account. Go to google.com, click sign in. Below the sign in box there is a link "Create an account now", click and create an account. Use this to sign in, check out the blog, and post questions/comments.
• Wait for an intern or another student to reply to your question. (Interns will check the student responses for accuracy)
• Check back to see a response.
• Still don't get it? Follow up with another question!

Easy as pie! Let's get started!

TAs and Professors are scary! Interns...Not so much.

Welcome to the BIOL3810 Molecular Cell Lab Intern Blog!

Hi, I'm Donna Burke.

Lab Intern Extraordinaire, Fall 2009!

Why I created this:

I, like most college-age students, have a fear of TAs and professors. I hate going to talk to them and I hate asking questions. They are smart, I don't want to waste their time with my questions that seem insignificant. This is why I created this blog, so students can talk to their peers, the interns, and get answers to the questions that seem small but are important to learning the material.

So here it is, the Molecular Cell Intern blog. This is for you, the students, to ask anything you want about Molecular Cell Lab! But as always, there are a few ground rules.....

Rules for the Blog:

1. This blog is for BIOL3810 Molecular Cell Biology Lab ONLY!
2. No homework answers, current lab reports, previous lab reports, past semester work, past semester exams, etc. will be tolerated. This is cheating and that's not cool.
3. There will be NO whining about TAs, Professors, Interns, Classmates, Classwork, Lab Reports, or CTW. We can't change these...get over it.
4. No ignorant or rude comments about questions or answers!! This is a happy place, a place for answers, this is not a place for mean spirits.
5. Ask whatever you want, as long as it pertains to lab and the lab content!

Please remember, Interns are not gods of Molecular Lab. They did not write the lab protocols and are not experts in this field. They are here to help you understand the protocols, lab results, writing reports, and the basics of the lab. They are just undergrads, too!