Lecture 3 - Handling and Electrophoresis Flashcards
what are the 5 things that affect the stability of a DNA duplex
length and base sequence+concentration
temperature
salts presence and conc
pH
denaturants
how can the pH of a buffer be checked before it is used in DNA analysis
using a pH probe
what is the beer lambert law and how can this be used in DNA analysis
A = ecl
A = absorption in nm
e = absorptivity = a standard value in M-1cm-1
c = concentration in microgram/ml
I = path length in cm
allows the concentration to be worked out based off the absorption of a solution which can be measured
how is the absorbance of a solution found (2 ways)
1 = can be measured by a detector
2 = can be calculated using the equation A = log (P0/P)
where
P0 = intensity before
P = intensity after
transmission = the same but without the log
how can the absorptivity if DNA vary slightly
at what wavelength does DNA often heavily absorb at and why
with the presence of single and double stranded molecules also a difference is seen with RNA
260nm due to the aromatic rings in the nucleobases
give three instruments that can be used to measure the concentration of DNA and 3 features about each of them
UV-Vis Spectrometer
- requires 1-3ml
- only one sample at a time
- gives whole spectrum (good for unknowns and mixtures)
UV-Vis Plate reader
- requires 50-300 microlitre
- lots of samples at once (used well plate)
- can give whole spectrum or single wavelength
(good for DNA analysis)
NanoDrop
- 1 microlitre
- one sample at a time
- normally only gives single wavelength
(can be too sensitive sometimes)
what theory does electrophoresis rely on
the fact that opposite charges attract and this can be done in solution to separate DNA
which direction does DNA migrate in electrophoresis and why
towards to +ve electrode (anode) as DNA has an overall -ve charge and oppsites attract
what defines the movement of different DNA fragments when electrophoresis is done in solution
therefore, what can be used to separate DNA strands based on their length
the movement is proportional to the mass to charge ratio -
fragments with the same ratio will move at the same speed in solution no matter their length
the use of gels acts as a selective medium in electrophoresis and allows different lengths to be separated
what are gels used in electrophoresis made of and how does this facilitate separation of DNA
intertangles fibrils and solvent stacks
smaller molecules are likely to move fast because:
- the fibrils can restrict the movement of larger molecules
- compacted DNA will move quicker than a rigid double helix
what does gel electrophoresis separate DNA strands based off
their size and shape as the mass to charge ratio is constant
which strands are found a the bottom of the gel
the shorter strands with smaller number of base pairs are visualised at the bottom of the gel closer to the +ve end
in electrophoresis what is used to compare a sample to a known length of DNA
a DNA ladder = the control run along side your sample so it is under the exact same conditions
what can be done to make an accurate estimate of the length of your DNA sample
the migration distance can be calibrated to the DNA ladder
what are the two types of gel commonly used in DNA electrophoresis
what is their
a) superstructure
b) casting method
c) typical gel conc
d) DNA size range
agarose (1) and polyacrylamide (PAGE) (2)
both of these can be native or denatured
1
a) tangles fibrils - physical gel
b) heat then cool
c) 0.5-2.0%
d) 50-30,000 base pairs
2
a) chemically cross linked matrix - chemical gel
b) radical polymerisation
c) 3.5-20%
d) 6 - 2,000 base pairs
