PLNT2530 PLANT BIOTECHNOLOGY    Winter 2023

Lab: Genomic DNA

This assignment is worth 30 points.

Due date: Wed. April 5

Goal: To extract genomic DNA from tobacco, and understand each of the steps.


1. Read through the protocol in lab manual:
Lab Manual - GenomicDNAManual.pdf

To make it easier to carry out the procedure in the lab, the steps described in the lab manual are condensed into a worksheet, which also has blanks for filling in information that cannot be done in advance. GenomicDNAWorksheet.xlsx.
In place of the Spectrophotometric method in the manual, we will use the Nanodrop device to quantify your DNA. We will also not be doing the PCR assay.
2. Watch the following videos which go into more depth about some parts of these procedures:

Lab Report Template
LibreOffice (GenomicDNATemplate.odt)
Microsoft Word (GenomicDNATemplate.docx)

Create a folder called GenomicDNALab to contain all files related to this lab eg. you might call it "GenomicDNALab". Save a copy of the Lab Report Template file to this folder.

Note: For your report, we will NOT use the Discussion questions on page 6 of the lab manual.

1. (18 points) DNA purification

Record your results on the worksheet provided. Typically, you will have a paper copy of the worksheet to be filled in during the lab. When complete, transfer your results to the spreadsheet file, which will be handed in along with your report.


NEW

Also in part 1,  include in your report the photo of the gel below (from 2022). What do the results show, versus what we should expect to see? If results were unexpected, explain what why they are different, and what might be a possible problem causing the observed results?
  GenomicDNATueGel.anno.png


Experiment numbers and labeling
Lab workers often overlook the need for precise labeling of materials such as tubes, boxes, filters etc. For any sample in the lab, it should be possible to unambiguously determine who produced it, what components and steps went into producing it, and when it was produced. The problem we face is that many of the materials we work with such as microfuge tubes, are small, allowing very little space for writing.

One solution is to give each procedure we do in the lab a unique experiment number. For PLNT2530, we will use the following scheme.

The Genomic DNA lab is the sixth lab in the term. The experiment number will therefore consist of your initials and the number '6'. For example, Dr. Fristensky's experiment number for this lab would be "BF6". All tubes would be labeled with the experiment number, plus additional information to describe the contents of the tube. By going to the worksheet for that experiment, you can find out what each tube is, based on the label.

sample
 label
This is the final purified tobacco DNA in TE. The concentration is added after Nanodrop quantitation. γ/λ is a common abbreviation for µg/µl
 BF6
tobacco DNA
[0.49 γ/λ]
negative control for EcoRI digest
 BF6
(-)
EcoRI digest
 BF6
EcoRI

When filling out the worksheet, make sure to put your experiment number on the worksheet, as well as on the tubes.

2. (12 points) Purpose of Purification steps

Any biochemical purification can be thought of as a succession of steps that separate classes of molecules from each other, based in different physico-chemical properties. When we grind open plant cells, the mixture contains macromolecules such as DNA, RNA, protein, carbohydrates and lipids, as well as small metabolites and clumps of insoluble cellular material (eg. cell wall material).

The flow chart at right gives a simplified representation of the genomic DNA isolation protocol.

For each of the following, state which components you think are in the different phases:
  • aqueous
  • interface
  • organic
  • pellet1
  • supernatants
  • pellet 3
Very briefly state your rationale for each choice.

If you wish, feel free to search for authoritative sources that indicate how these different components partition into different phases. Make sure to provide references if you use outside sources.

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