PCR Lab Home
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Nic Theobald Yilectronics
Introduction:

The goal of this lab was to perform PCR on E. coli bacteria in order to make multiple copies of a DNA segment. With the these DNA segments, gel electrophoresis and/or DNA sequencing can be used to determine the number of base pairs in the DNA segments. It also demonstrated proper lab technique such as pipetting, aseptic technique, plate streaking, inoculation, etc. This tutorial was intended to be followed linearly, but the reader is welcome to jump around as needed...

These tutorials will be structured in a manner that presents background information about the topic and then the procedures that were actually performed during the lab (Lab Procedures). Several of the topics below will have a seperate page with more information and videos.

Note: I hope to recreate the effectiveness and approach of this lab, but will heavily lean on outside youtube videos to teach proper technique. Sources for all outside content can be found at the bottom of their respective pages and at the bottom of this page.

Lab Techniques and Culturing:


In the effort of maximizing repeatability, it is of the utmost importance that you are consistent and follow proper procedures. The following page will cover several topics including aseptic technique, pipetting, inoculation, and plate streaking. The techniques discussed on the page and in the videos will be important for further procedures.

PCR Background:

The Polymerase Chain Reaction (PCR) is a technique used to make many copies a specific segment of DNA. The video below shows how the PCR process works on the molecular level.
 



Benchling and The National Center of Biotech Information (NCBI):

Benchling is a tool used for DNA sequence editing, design, and analysis. This tutorial will demonstrate how to determine what primers you need in order to isolate a specific segment of DNA.

Gel Electrophoresis.

Gel electrophoresis is the process of separating DNA base pairs by size. Using this technique, you can approximate which base pairs are present in the sample and verifiy that the PCR process isolated the correct segment.



DNA Sequencing:

DNA sequencing is the process of determening the exact sequence of base pairs. The PCR product in this lab was prepared to be sequenced, for practice, but was not actually sequenced.



Cell Storage Techniques:

Living cells need to be refrigerated in order prevent further multiplication. If left in a warm environment, cells in a culturing media will start to form one or many large colonies and make it difficult or impossible to isolate one colony for study.

Liquid and plate cultures can be stored in the refrigerator at 4C for several months. When storing plate cultures in the refrigerator, wrap the edges of the top and bottom with stretch tape. However, the culture media will slowly evaporate and the colonies will die off. For long term storage the cells can be cryogenically frozen at -80C.

Further guidance would be required for the actual freezing and storing process, but here is how you would prepare the cultures for freezing:

Creating 45% Glycol Mixture

  • Slowly pipette 450 microlitres of glycerol into a small test tube (it may take fractions of a minute just to draw the glycerol into the pipette)
  • Pipette 550 microlitres of deionized water into the same small test tube
  • Agitate the test tube until the viscous glycerol is completely mixed with the water
Preparing Cryovials
  • Slowly pipette 1 milliliter of the Glycol mixture and 2 milliliters of the liquid culture into the cryovials
  • Label the cryovials
  • Further guidance would be needed for the rest.

Lab Procedures:


Living cells need to be refrigerated in order prevent further multiplication. If left in a warm environment, cells in a culturing media


Page created by Nic Theobald 8/9/2020
Last Update: 9/17/2020