DNA and RNA gels Flashcards
What is the process of getting DNA out of a sample?
Lysis
Protein removal
Precipitation
What is lysis of DNA?
Breaking down the sample using a pestle and mortar.
Place in a buffer - Tris HCl, which maintains pH.
EDTA is a chelating agent which inhibits the nucleases so DNA and cell remains intact.
SDS detergents dissolves the lipid membranes.
What is protein removal from DNA?
Saturated NaCl helps removal proteins that are bound to DNA.
It also keeps the proteins dissolved in the aqueous layer so they can be removed by pipetting.
What is precipitation of DNA?
Ethanol is added to precipitate the DNA, so it can be spun in a centrifuge.
The supernatant containing the protein is removed, and the pellet containing the pellet is kept.
DNA is then rehydrated using a solution of water.
How are pure samples of nucleic acids quantified?
A spectrophotometer measures the amount of ultraviolet radiation absorbed by the bases.
Starts at wavelength of 220nm, and peaks at 260nm for nucleic acids.
How is a spectrophotometer used?
H2O or 1X TE is used as a solvent to suspend the nucleic acids.
The sample is then placed in a cuvette, which is 1cm thick.
The spectrophotometer is set to zero using a sample of solvent.
How do you calculate the amount of nucleic acid?
The values for a 1cm pathlength will be the same, so the optical density for the set type of nucleic acid will be the same (dsDNA, ssDNA, oligonucleotides, and RNA).
So times optical density by the concentration = concentration of DNA in sample in ug/ml.
Then do 1/this number to convert the units to ug to find the amount.
How can the purity of the nucleic acid be determined?
Calculate the OD260/OD280 ratio.
On a graph, there will be a double peak which shows contaminated with proteins.
What are the ratio purity values of the nucleic acids?
Pure DNA has a ratio of 1.8.
Pure RNA has a ratio of 2.
A higher ratio means the nucleic acid hasn’t been purified well.
How can DNA and RNA be quantified using fluorescence?
Measure the UV-induced emission of fluorescence from intercalated dyes - picogreen or ethidium bromide.
The dye binds to DNA and emits wavelength.
The more dye bound, the higher the wavelength.
Used if the amount of DNA is not enough for a spectrophotometer or is contaminated.
What is gel electrophoresis used for?
The sizes of DNA/RNA/protein fragments.
The purity of these samples.
The condition of these samples e.g. degradation.
What are the DNA and RNA gels?
Agarose or polyacrylamide.
What is the set up for gel electrophoresis?
Weigh out agarose.
Place in a buffer - TAE or TBE.
Heat to ensure all agarose is in solution.
Pour into a casting tray.
Place in a comb.
Allow to set.
What is the basic principle of gel electrophoresis?
Uses electric charge to draw the samples through the gel, and separates the DNA or RNA.
The distance things travel depends on charge, shape and size.
What is the concentration of agarose solution?
The more agarose in solution (higher percentage), the tougher and more concentrated the gel, and the smaller the holes in the matrix will be.
How are DNA samples loaded into the gel?
DNA samples are pipetted in, using a loading buffer.
The buffer contains bromophenol blue, to see where the sample is, and glycerol.
Glycerol makes the sample heavier than the buffer so it sinks to the bottom of the well.
The well is shiny because it contains a TAE or TBE buffer.
What else is bromophenol blue used for?
Bromophenol blue and xylene cyanole dyes migrate through agarose gels at roughly the same rate as dsDNA fragments .
This is so you know when to stop the charge so the DNA does not travel too far.
What does DNA do in gel electrophoresis?
DNA runs from the cathode to the anode (+) because it is negatively charged, and separates out.
The intercalating dye - ethidium bromide or gel red, binds to the minor grooves and emits fluorescence under UV to visualise the DNA.
What is a DNA ladder?
It is used to know how big the DNA is.
It tells you the molecular weight.
It has 2 wells of DNA of known size to compare to.
Different DNA ladders are used depending on the estimated size of DNA - low range to high range.
How does the percentage agarose change gel electrophoresis?
The % concentration per volume changes how quickly and easily DNA is able to migrate through gel.
Larger DNA fragments use a lower % so they move quicker through the gel.
Small DNA fragments use a higher % to slow the DNA down and be separated out.
What is the relationship of migration distance?
Migration distance is inversely proportional to the log10 of the molecular weight.
Large fragments migrate less, small fragments migrate further.
1/Log10 MolWt
How does voltage affect the mobility of DNA fragments?
As voltage increases, larger fragments migrate proportionally faster than small fragments, as they have more charge.
How does the electrophoresis buffer affect the mobility of DNA fragments?
Establishes pH
Provides ions so charge moves through
Different ionic strength - so can run a higher voltage.
TBE is a stronger buffer - so higher voltage and longer runs.
TAE is used for linear DNA.
How does closed DNA migrate in agarose?
Uncut plasmids will appear to migrate more rapidly that the same plasmid when linear.
Closed circle will go through holes in gel faster than if cut with double stranded endonuclease and fragment of DNA on its own.
How does the shape of DNA differ migration?
Supercoiled DNA will migrate easier through the matrix gel compared to open or closed circular DNA.
Linear double stranded DNA migrates easily.
Single stranded DNA is more flexible, so migrates faster.
What is the order of DNA migration?
Supercoiled (most compact, fastest)
Linear (moderate speed)
Relaxed Circular (less compact, slower)
Nicked Circular (least compact, slowest)
How does DNA agarose gels show the purity of DNA?
Smears of DNA down the column shows the DNA has started to break down, so are not in the same size, and shows the DNA is poor quality.
Proteins cluster at the top of the gel which shows low purity.
It can confirm restriction enzyme digests.
What are DNA acrylamide gels?
Separates very small DNA fragments, usually oligonucleotides.
Contains the non-ionnc denaturing agent urea, which prevents secondary structure formation of the oligonucleotides binding to themselves.
Accurate determination of molecular mass - 1 base difference.
What is RNA gel electrophoresis?
Agarose gel contains formaldehyde as a denaturing agents, which limits secondary structures.
This is because folded RNA does not evenly migrate through the gel.
However, formaldehyde is toxic by skin contact and inhalation of vapour.
What is the principle of RNA gel electrophoresis?
Same principal and properties as DNA.
The loading buffer contains MOPs, formaldehyde and ethidium bromide.
What is MOPs?
MOPs is able to maintain the buffering capacity at neutral and lower pHs compared to Tris based buffers.
What is the results of RNA on denaturing agarose gel?
Common RNA is rRNA, so there will be clear 28s and 18s bands, which can be used to check how good the RMA sample is.
If RNA is degraded, everything will be a lower weight smear.
What is RNA on a non-denaturing agarose gel?
Looks at the structures created in the cell, not just the RNA.
Uses the same agarose gel but no formaldehyde is added, only TBE or TAE, and bromophenol blue or xylene cyanole.
Can distinguish complexes and different folded forms of RNA.
What is RNA denaturing polyacrylamide gel?
Has very good resolution - one base difference.
Excellent for sequencing.
Following the restriction enzyme digestion the student must prepare a 1.6% Agarose gel (FW120) diluted in TBE from a 20x TBE Stock to cast a 150ml gel.
Calculate the amount of agarose and TBE buffer required to cast the gel
Agarose 1% = 1g/100ml
1.6% = 1.6g/100ml
1.6 x 1.5 = 2.4g/150ml
TBE buffer is at 20x so do 1/20 dilution
C1v1 = c2v2
20 x V1 = 1 x 150
= 7.5ml 20x TBE
c. The resulting ethidium bromide stained gel (shown below) indicates that the computer- generated map is in error. In particular, one of the predicted ClaI sites is not present in the DNA fragment and represents a sequencing error. Why do you know this?
There are fewer fragments than expected.
The distance between the fragments align to the bands, so can tell which fragment is missing.
see picture on SGT.
