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# System prepended metadata

title: PCR

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# PCR

[EcoEvoDevo Lab](https://hackmd.io/@aphanotus/AngeliniLab)

![](https://i.imgur.com/J61byHq.jpg)

## General considerations

### Be organized

You will likely undertake many similar reactions in your time in the lab, so being able to identify individual reaction products in a freezer box months later will be important. Keep careful records in your lab notebook. (Refer to the SOP on lab notebooks.) Always give a brief explanation of why you're doing each procedure and what you hope to achieve from it. Critically, your notebook must be dated and list the ID numbers of the samples you generate. 

The standard method our lab uses for identify samples created through molecular methods, like PCR, is to use your initials, a unique number, and the date. So, the first reaction I generate today, May 30, would be "DA#1/5-30". It is also important to provide some explanation of what the sample is. PCR products can go in a box labeled "PCR products" in the refrigerator (4˚C). But other, less common sample types should be labeled appropriately. For example "Of total RNA", "Jh L5 male cDNA", etc. If a concentration is measured, that value should be recorded in your notebook and on the side of the sample tube.

Make note of the expected size of the PCR products you are intending to generate. Typically, after a PCR run, the size of the products are confirmed using [agarose gel electrophoresis](https://hackmd.io/@aphanotus/agarosegels).

## Diluting new PCR primers

New oligonucleotide primers will arrive freeze-dried in individual tubes. The datasheet provided by the vendor (typically Sigma-Aldrich) will list the number of picomoles or (easier) the volume to be added for a 100 μM solution. 

- Label the white cap of the tube with the primer name.
- Reconstitute a 100 μM stock solution using 1X T E buffer. Add the volume of TE indicated on the datasheet. Be sure the freeze-dried pellet is not up in the cap.
- Cap and vortex the tube.
- Incubate at 55˚C for 5-10 minutes on a dry block.
- Vortex.
- To prepare a 10 μM working concentration of the primer, transfer 10 μl of the primer stock into a new tube with 90 μl of nuclease-free water. 
- Store the primer stocks and working dilutions at -20˚C. 
- Organize primers into freezer boxes for primers arriving within a certain dated period or dedicated to a particular project.
- Save the oligo datasheets in the Oligo binder.

## PCR

The polymerase chain reaction (PCR) is a method for in vitro DNA synthesis using specific primers. PCR can be used to make billions of new DNA molecules that are copies of one specific sequence from a complex mix of DNA molecules. PCR is one of the most important and commonly used methods based on molecular genetics. The reagents need for PCR include the following.

- Taq DNA polymerase is the enzyme that builds new DNA molecules. 
- Buffer provides Taq with the pH and salt concentration at which it works best. 
- dNTPs (deoxynucleotide triphosphates) are the monomers that will be combined to make DNA. 
- Template DNA must be present to be copied (or "amplified"). Depending on your application, you may use genomic DNA, cDNA, or another PCR product as template.
- Primers are short (10-50 bp), single-stranded oligonucleotides that determine the sequence to be amplified. Two primers must be used—one for each of the two template DNA strands.
- Water is added to make up the remaining volume.

During a PCR reaction, the solution is heated to separate (or “denature”) the two template DNA strands, then it is cooled slightly to allow the primers to base-pair (or “anneal”) at the appropriate sequences on the template. Finally, the temperature is adjusted to 72˚C, which is optimal for Taq to extend the primer, adding new nucleotides to the 3’-hydroxyl, and creating a new complementary DNA strand. These steps are then repeated 30-40 times, allowing the products of early cycles to act as new templates in later rounds. The result is exponential growth in the number of DNA molecules with nucleotide sequence determined by the original template sequence between the two primers. 

As you work, be mindful that you do not unintentionally cross-contaminate your reagents. Once a pipette tip has been used, it should be discarded. Keeping your reagents and samples cold will help preserve perishable molecules like dNTPs and prevent your reaction from starting prematurely, before primers can properly bind to the template.

### PCR reaction set-up

Our lab typically uses a mix of Taq, Buffer and dNTPs called [JumpStart](https://www.sigmaaldrich.com/catalog/product/sigma/d9307), made by Sigma-Aldrich. The typical reaction size is 12 μl, which provides 6 μl to run the product on a gel and 6 μl left for downstream applications. In certain circumstances you may need to scale this size up.

- For a small number of reactions, you can use single 200-μl PCR tubes. For many reactions, use "strip-tubes" which link 8 tubes together.
- Label your reaction tubes with your initials and the date. Number multiple tubes.
- Each PCR reaction should include the following reagent volumes

|        |                    |
| ------:|:------------------ |
| 6.0 μl | JumpStart Taq Mix (2X)  |
| 0.5 μl | forward primer  |
| 0.5 μl | reverse primer |
| 0.5 μl | template (e.g. cDNA) |
| 4.5 μl | water (nuclease-free) |
| 12.0 μl | total |

- If reagents appear on the side of your tube, spin it briefly in the mini tabletop centrifuge, using the adapter for 0.2 ml tubes.
- Mix the contents of your tube by pipetting up and down. The volume setting is not critical, and can be anywhere from 5-10 μl. Keep the pipette tip below the surface of the solution, to avoid introducing bubbles.
- Close the caps on your tubes tightly to avoid evaporation.

#### Master mix

If you are setting up multiple reactions, it is convenient to create a "master mix" by multiplying the volume of reagents that are common to all reactions by the number of reactions. 

For example, if you are using the same template in 8 reactions, but each uses different primers, then mix 48 μl JumpStart stock, 4 μl template, and 36 μl water. After mixing, move 11 μl of this master mix into 8 tubes. Then add 0.5 μl of the appropriate primers to each tube.

Once all the reagents are added to a reaction, be sure it is mixed by pipetting up and down. For large numbers of reactions, store your tubes on ice to keep reagents cool as you assemble all the reactions. 

#### The thermocycler and PCR programs 

Our lab uses a BioRad C1000 Touch Cycler with dual 48-sample gradient heat blocks. This machine can run two programs at once and can be used for PCR or any complex thermocycling application.

While you may want to vary the conditions of your PCR reaction (see below) a standard PCR thermocycling program looks like this.

|      |        |              |
| ---- |:------ |:------------ |
| 98˚C | 2 min  |              |
| 98˚C | 10 s   | \|           |
| 55˚C | 30 s   | \| 35 cycles |
| 72˚C | 30 s   | \|           |
| 72˚C | 2 min  |              |
| 12˚C | hold   |              |

This program will take about 90 minutes to run. When the reaction is finished, the samples will be held at 12°C. 

- Within 18 hours, cancel the run and move your PCR products to the refrigerator. The PCR machine is not designed to sustain low temperatures for long periods of time.

Depending on the primers you are using you may want to adjust the parameters of the PCR program.

- The annealing temperature can be adjusted depending on the predicted Tm of your primers. Lower annealing temperatures are more permissive; higher temperature are more stringent. So a lower annealing temperature may produce more bands: the intended product as well as non-specific products. The feasible range for annealing temperatures is 40-65˚C.
- The extension time can be adjusted depending on the length of your intended PCR product. Taq polymerase works at a speed of 1kb / min at 72˚C. So, adjust the time accordingly.
- The number of cycles can also be adjusted. If your PCR produces few bands, you can increase the number of cycles. This can work for reactions with very low copy-number templates.

> Some other tips for trouble-shooting hard to amplify reactions.
> - Try, try again.
> - Try multiple annealing temperatures. Up to 8 can be tested using the gradient block feature.
> - The quality of template makes a big difference. Is your template good? Try a positive control.
> - A series of reactions using nested primer pairs is often successful for degenerate PCR.
> - MgCl~2~ can be added to the reaction to decrease stringency of the primer binding without adjusting temperature.

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[EcoEvoDevo Lab](https://hackmd.io/@aphanotus/AngeliniLab)

