1. DNA Extraction from Living Cells Flashcards
(35 cards)
All cells capable of dividing to generate two daughter
cells contain what
genomic dna
what is genomic dna
a complete set of chromosomes which makes up the blueprint for those cells
is dna easily extracted
in plants and animals yeah
Isopropanol is an alcohol- safety thing?
avoid breathing in its vapours
yes flammable
SDS and proteinase K will affect what parts of the body
skin, causing irritation and eyes (can severely damage)
Where is DNA located in the cell?
DNA is primarily located in the cell’s nucleus in eukaryotic cells (like plant and animal cells). In prokaryotic cells (like bacteria), DNA is located in the cytoplasm, usually in a region called the nucleoid.
What other nucleic acids are present in cells?
Besides DNA, cells also contain RNA, which is involved in protein synthesis. Types of RNA include messenger RNA (mRNA), transfer RNA (tRNA), and ribosomal RNA (rRNA). Some cells may also contain small amounts of other types of RNA, like microRNA.
What other components do we need to separate the DNA from?
In addition to DNA, cells contain proteins, lipids, carbohydrates, and RNA. The purpose of DNA extraction is to isolate DNA from these components, particularly proteins (which may need to be broken down) and lipids (which need to be removed to prevent contamination).
What is the purpose of homogenisation?
Homogenisation is a process used to break open the cell membrane and other cellular structures to release the DNA into a solution. This process helps to ensure that the DNA is separated from the other cellular contents, making it easier to extract and purify.
What is a standard curve?
A standard curve is a graph used to determine the concentration of a substance (like DNA) in a sample by comparing the sample’s measurement (e.g., absorbance at a specific wavelength) with a set of known concentrations. The standard curve allows you to estimate the amount of DNA in your sample based on its absorbance.
what machinery do we use to homogenise
Rotor-Stator Homogeniser
method for preparing the homogenate
- slice large onion
- sufficient extraction buffer added to cover the diced onions in a 500 mL beaker (200 mL total).
- onions homogenised in machine for 1-2 mins
- then filtered with a sieve
- tissue is then homogenised with a further 200 mLs of buffer and filtered.
This gives about 300 mLs of filtered homogenate for a class of 50.
what plant do we use sample and what for the standard for dna extraction
onions for DNA extraction, and
commercially available Herring sperm DNA is provided
for standards
what is the extraction buffer
(0.1 M NaCl, 50 mM Tris pH 7.5)
method of the practical: what do we after we take e.g. 5mL of the prepared homogenate
. Add 0.5 mL 10% SDS (sodium dodecyl sulphate
detergent).
- tighten the lid, mix by gently inverting the tube
two or three times.
Describe the changes from that you can
observe over 10 minutes
When you add 0.5 mL of 10% SDS (sodium dodecyl sulfate) to a homogenised onion sample and gently mix, the following observable changes are likely to occur over the next 10 minutes:
Immediately after adding SDS and mixing:
Increased cloudiness or opacity: The solution becomes more turbid as the SDS begins to lyse cell membranes, releasing cellular contents.
Foaming or bubbles: SDS is a detergent, so gentle mixing may produce a foamy layer at the top.
Over the next 10 minutes:
Solution appears more uniform: The solid fragments may seem to dissolve more into the liquid, as SDS breaks down membranes and nuclear envelopes.
Viscosity increases slightly: As DNA is released into the solution, it may become slightly thicker or more syrupy.
No major colour change, though a slightly more yellow or cloudy tone may develop.
why does SDS have this effect
SDS disrupts phospholipid bilayers and denatures proteins, effectively lysing the cells and nuclei, which helps release DNA into the solution.
what do we add after sds and why
note: we again tighten the lid, mix by gently inverting the tube two
or three times, after adding this
Proteinase K after SDS to digest the proteins that are released when cells are lysed.
By removing proteins and enzymes, Proteinase K helps ensure the final DNA sample is clean
finally, we tilt tube to 45° and gently run propan-2-ol
down the side of the tube using a bulb-pipette - why do we add this?
so that two layers are formed.
Take Care! If done too quickly the layers will mix and
you will not get any DNA!
For DNA quantification: 2. Transfer the DNA sample from the eppendorf tube to a UV spectrophotometer cuvette using a pipette.
- Use the same cuvette for each sample: why?
different cuvettes have different intrinsic UV absorbances
What is plotted on the standard curve in DNA quantitation? (x and y axis)
Y-axis: A260-A320 absorbance values; X-axis: DNA concentration (μg/mL).
Why is the DNA measured at 260 nm first?
DNA absorbs UV light strongly at 260 nm, which allows quantification of DNA concentration.
Why is absorbance also measured at 320 nm?
320 nm detects background absorbance or turbidity; it corrects the reading by removing non-DNA interference.
cDNA in bioinformatics refers to an mRNA transcript sequence expressed as DNA or RNA bases?
as DNA bases
