BMP: EXP5 electrophropheris DNA Flashcards
What is technique is used to seperate DNA fragments? What is this based off?
elecrophophresis - size of fragments
What matrix is used?
Highly purified agar gel
Describe what happens during the electropophresis of DNA
When an electric field is applied to the argrose gel in the presence of a buffer solution the DNA charge fragments migrate from te -ve to +ve end due to the net -ve charge of the DNA phosphate back bone. Seperation based upon nucleotie size is effected
Describe the requirements of the argrose gel and how changing concentration effects the electrophophresis
The agarose gel matrix needs to contain a highly purified matrix of agar gel, which seperates DNA molecules dependent upon DNA molecule SIZE.
Different concentrations of agarose gel can be used. Increasing concentration of agarose gel creates a sieve with smaller pores, which makes larger DNA molecules move more slowly than smaller ones
WHat is the relationship betwen size and distance travelled by the molecules?
Why is DNA negatively charged?
- LINEAR- The distance travelled by the DNA molecule is inversely proportional to the logarrithm of the molecular weight
- Circular (plasmids) - When DNA molceules are circular they are composed of 2 bands. The lower band is composed of negatively supercoiled plasmids. The DNA is very compact so is more mobile. The lower band is assoicated with open circular DNA or nicked DNA that is formed from the super-coiled DNA by breackage of one of the stands
There is phosphate groups joining the nucleotides together, the OH is ionised leaving O ions therefore negative
What DNA base is found in DNA but not RNA
thymine
How many hydrogen bonds occur between ACGTU
CG - 3
AT - 2
AU - 2
What happens to the DNA prior to application to the gel?
It is mixed with a loading dye containing sucrose and a one or mole coloured dyes.
- Sucrose is used to add density to the sample, keeping it in the well when applied.
- Dyes - act as a visual marker, which acts as an aid to the monitor progress of the electrophoresis
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How does visulisation of the DNA frgaments occur?
Visulisation of DNA fragments after electrophoresis is acheived using gelred.
The gel is soaked in a dilute solution of gel red after electrophoresis or gel red is incoperated into the gel prior to electrophoresis.
The gelred binds to DNA producing a comlpex that fluoresces when exposed to UV light
The red gel forms a complex with the DNA which fluoreses which exposed to UV
How could a permenant record be kept of the electrophophersis sample?
Photograph it or use specially desgined softwarefor it
- How does voltage effect electrophoresis
- What do you need to be careful off?
- INcreasing the voltage increases migration distance. Increasing duration increases migration distance.
- Too high a voltage or too long may lead to DNA moving off the end of the gel into the buffer solution
**not question - just diagram off electrophoresis equipement**
How doe analysis of the bands occur during visulisation?
Gel red stains nucleotides
has an excitaory peak at 300nm which can be excited by a common 300nm uv transilliminator
Has an emission peak at 395nm which is compoatabile with ethidium bromide filter or SYBR filter
What visulisation technique is preferred? why?
gel red as safer and more sensitive
ethidium bromide is geno toxic
After visulisation of the sample has occured how are reuslts determined?
- Distances travelled by samples compared to marker DNA fragments of known size
- Meausre the distance from the edge/ middle of the well to the middle of each band
- PLot a graph of log molecular weight (in base pairs) - y axis against distance migraed (mm) - x-axis
- Use the distanc emigrated for unknown and interpolate using the graph to determine unknown size
