Name:___________________________________

Fish 310 Spring 2015

#Quarter Project Lab D

Supplies:

- cDNA from Samples
- Ssofast Evagreen qPCR Mix
- Nanopure H20
- Primers
- qPCR plates
- qPCR machine
- Strip Caps
- Pipettes
- pipette tips
- 1.5 ml tubes
- Ice buckets
- Ice

Agenda:

- Perform qPCR
- Presentation
- Discuss groups data
- Collect data from qPCR

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#Differences in Olympia Oyster Populations at the Gene Expression Level

As part of your experiments you are comparing how different populations will respond to stress to confirm your hypotheses that were generated as part of "Lab A".  This graph should ring a bell.

![death](http://eagle.fish.washington.edu/cnidarian/skitch/course-fish310-2015_QP-lab-A-worksheet_md_at_master_·_sr320_course-fish310-2015_1AFD6852.png)

Today what you will be doing is learning how to measure differences in gene expression. This is a _very valuable_ core technique in biology and works on the general principles of the Polymerase Chain Reaction. The polymerase chain reaction (PCR) is a technology used to amplify a single copy or a few copies of a piece of DNA  generating thousands to millions of copies of a sequence. 

![PCR](http://eagle.fish.washington.edu/cnidarian/skitch/pcr_1AFD6978.png)
 by Enzoklop
 
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In our case we will be using PCR a fancier version of what is shown above in the sense that we are interested in quantifying the level of gene (mRNA) expression (so we have to make cDNA first) and a flourescent dye is added to the reaction so we can measure easily. 

**Biology Refresher**
![dogma](http://eagle.fish.washington.edu/cnidarian/skitch/transcription_and_translation_1B00E632.png)

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_Reverse Transcription_
![rev-tran](http://eagle.fish.washington.edu/cnidarian/skitch/cdna_-_Google_Search_1B00E717.png)

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_SybrGreen_
![sg](http://eagle.fish.washington.edu/cnidarian/skitch/quantitative_pcr_sybr_green_-_Google_Search_1B00E7D1.png)


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_qPCR Results_
![curve](http://eagle.fish.washington.edu/cnidarian/skitch/quantitative_pcr_-_Google_Search_1B00E867.png)

_Tip_  You can use the following formula to convert Ct values.     
Arbitrary expression value =10^(-(0.3012*Ct)+11.434)

## Quantitative Polymerase Chain Reaction

You will be setting up your own PCR reactions with cDNA that was made from RNA isolated from the tissue samples you took last week. The recipe is below, with `Ssofast Evagreen MM` being a premix that contains nucleotides, buffer, DNA polymerase, and SybrGreen flourescent dye. The primers are specific to the gene you will be measuring (see PCR figure above). H20 is used to make the volume appropriate (think concentration). Below is the recipe for one reaction and for 8.

|                     | Volume (ul) | Reactions (ul) X8 |
|---------------------|-------------|-------------------|
| Ssofast Evagreen MM | 10          | 80                |
| FWD Primer          | 0.5         | 4                 |
| REV Primer          | 0.5         | 4                 |
| Nuclease Free H2O   | 8           | 64                |



1. Add each volume (REACTIONS (ul) X8) of reagents in order from greatest to smallest REACTION in 1.5 ml tube (Master Mix)
2. Pipette 19 ul of master mix to each of the 7 tube wells
3. Pipette 1 ul of water into 1 tube
3. Pipette 1 ul of Control into 1 tube
4. Pipette 1 ul of each sample into each of 5 tubes
5. Added clear strip caps to tubes (DO NOT WRITE ON STRIP CAPS)
6. On a separate sheet of paper write down the order of your samples in the wells


##qPCR Questions
 

 

 

 

 

 

####What primer did you use? Why?
 

 

 

 

 

 

####How does this relate to the project?
 

 

 

 

 

 

####How does qPCR work?
 

 

 

 

 

 

####What do these curves mean?
 

 

 

 

 

 

 

 

 

 

####What do you expect to see from your qPCR run?
 

 

 

 

 

 

##Analysis questions
####What type of data do you have from the experiment?
 

 

 

 

 

 

####What statistics will you use to analyze your data? 
 

 

 

 

 

 

####How will you do your statistics (programming, by hand, etc)
 

 

 

 

 

 

####What other data might you need to help you analyze the information from the experiment?
 

 

 

 

 

 

####What graphs or other images will you use to represent your data?