441_Lab5_2013


 * Lab 5: P **** rep for Experiment, primer design, primer reconstitution, and PCR **


 * // Please print these lab materials before each lab. //**


 * Lab Objectives **
 * Mock-up Experiment to prepare for the initiation of experiments
 * Design and order primers
 * Rehydrate, dilute primers so that they are ready to do PCR
 * Perform PCR on cDNA/gDNA with the primers you ordered


 * Experiment Mock-up **
 * 1) 1. Now that you know what your biological question is and what controls and replicates you'll need for this experiment, you'll need to do a mock-up of the experimental containers so that the system will be ready later in the week when your experiment starts.
 * 2) A number of containers, heating elements, water baths, airstones and pumps are available. Work as a group to decide how the experiment will be organized.


 * Primer Design **

Primers, or oligonucleotides (oligos), are short stretches of synthetic DNA that are used most commonly for PCR and DNA sequencing. They direct DNA polymerases to specific regions on larger DNA molecules for amplification. They are designed in pairs to amplify DNA in the forward and reverse directions. Oligos are custom synthesized by various manufacturer's to contain the precise sequence requested by the customer. For a good introduction to the theory of primer design procede to this [|link].

Here is a brief list of things to take into consideration when designing primers. Although none of these are absolute, they will help ensure your primers will hybridize to your target sequence with the best efficiency.

1. Design your primers to be within 18-30 bases in length.

2. The melting temperature (Tm) of primers should be within 2C of each other.

3. Avoid primer dimers and primer hairpins

4. Avoid high G/C stretches, particularly at the 3' end

5. G/C clamp at 3' end of primers.

Primer design is most commonly done via computerized means and the algorithms used take the above rules into consideration. Of course, the user always has the opportunity to adjust the parameters that define how primers are designed by the software. There is a great deal of software available for primer design. Two commonly used primer design tools are [|NCBI Primer] and [|Primer 3]. The software will allow you to enter a full DNA sequence and then define what region(s) you would like to amplify, the ideal size of the amplicon (PCR product), the ideal length of the oligos, etc. However, often the preset conditions are already optmized to Additionally, after you have selected some proposed primers, you can compare melting temps, G/C content, primer dimer/hairpin probabilitites, etc.

We will go over the process of designing primers in more detail in lab.

__** Primer reconstitution (to be explained in lab) **__ The primers you designed are in a dehydrated state. After re-hydrating them, we will be making 100uM stock and a 10uM working stock to use for PCR.
 * Polymerase chain reaction **
 * Supplies and Equipment: **
 * Micropipettes (1-1000 μl)
 * Sterile filter pipette tips (1-1000 μl)
 * Tip waste jar
 * PCR tubes (0.5 ml; thin walled)
 * 1.5 ml microcentrifuge tubes (RNAse free)
 * cDNA (student provided)
 * dNTPs
 * 2x GoTaq Green Master Mix
 * Primers
 * Nuclease Free water
 * thermal cycler
 * Kimwipes
 * microfuge tube racks
 * PCR tube racks
 * ice buckets
 * Lab coat
 * Safety glasses
 * Gloves


 * Procedure background **

The polymerase chain reaction involves selective amplification of a DNA (genomic or complementary) target using the enzyme polymerase (after which the method is named), primers (short oligonucleotides), and dNTPs (A, C, T, and G). The method relies on thermal cycling, which consist of repeated heating and cooling of the reaction for DNA melting and enzymatic replication of the DNA. These heating and cooling cycles are comprised of three primary steps. A single cycle begins with denaturation at ~94°C and during this step the DNA is melted or rather unwound and the strands are pulled apart resulting in single stranded DNA. This is followed by an annealing step at ~50-60°C where the primers anneal to the target sequence. After which there is an extension step at ~72°C where nucleotides DNA polymerase synthesizes a new DNA strand by adding dNTPs that are complementary to the template. As PCR progresses, the newly generated DNA is also used as a template for replication, setting in motion a chain reaction in which the DNA template is exponentially amplified. This process allows us to generate thousands to millions of copies of a particular DNA sequence with only a single or a few copies of a piece of DNA.

For this lab we will be using Promega’s Go-Taq green master mix, please read the __ manufacture’s protocol __.

You will be preparing 50 µl PCR reactions: count how many total samples you have ( = x), add two negative controls (you will use nuclease free water instead of cDNA template), and add one extra reaction to account for pipetting error. For example, if I have 3 cDNA samples, I will calculate my master mix for 6 reactions.
 * PCR PROTOCOL **

1. Make a reaction mastermix in a 1.5 ml microcentrifuge tube labled "MM" and with your initials. 2.Prepare your mastermix by first calculating the total volume required for each component of the mastermix, then pipette each reagent and mix well. The following volumes are what you would need for __a single__ reaction: 3. Pipette 48 ul of your master mix in to each of your 0.5 ml PCR tubes labeled with your sample name and with your initials 4. Add 2 ul of the appropriate template to each tube and mix via pipetting 5. Spin tubes to pool liquid at the bottom of the tubes. Load reactions into thermocycler making sure the caps are tightly secured. 6. Your samples will be put through the following thermal cycling profile and then stored at -20°C afterwards.
 * Reagent || 1.reaction || x.reactions ||
 * 5x GoTaq Green buffer || 10 µL ||  ||
 * 10mM dNTP mix || 1 µL ||  ||
 * 10 µM forward primer || 1 µL ||  ||
 * 10 µM reverse primer || 1 µL ||  ||
 * GoTaq polymerase || 0.25 µL ||  ||
 * nuclease-free water || 36.75 ||  ||
 * Step || Temperature || Time || Cycles ||
 * Denaturation || 95C || 5 min || 1 ||
 * Denaturation || 95C || 30 sec || 40 ||
 * Annealing || 55C || 30 sec ||^  ||
 * Extension || 72C || 90 sec ||^  ||
 * Final extension || 72C || 3 min || 1 ||
 * Hold || 4C || ∞ || 1  ||