MCG - (Practical Pt.2) Use of PCR to Genotype Individuals Flashcards
TRUE or FALSE?
Under the influence of the electrical field, the positively charged DNA will migrate towards the negatively charged end of the gel.
FALSE.
The statement is false because DNA is negatively charged, which will migrate towards the positively charged end of the gel.
Which is the most preferred buffer condition for agarose gel electrophoresis?
~7.2 pH
DNA will migrate through the gel towards the positive electrode since DNA has a strong negative charge at neutral pH.
Which of the following factors will not affect the rate of migration of DNA in agarose gel?
a) DNA concentration
b) Gel concentration
c) Voltage
d) DNA conformation
a) DNA concentration
Concentration of DNA will not affect the rate of its migration.
An increase in gel concentration causes the migration rate to decrease.
An increase in voltage causes the molecules to migrate faster through the gel. However, the resolution will decrease at high voltage and there is a risk of melting the gel due to the increased heat.
Different conformations of the same DNA affect the effective size of the DNA. For example, different forms of a plasmids will migrate at different rates - closed, circular forms migrate faster than linear forms. To avoid this problems, linear molecules are usually analysed.
If 2% (w/v) of agarose in gel is used, what is the effective separation size range of double-stranded DNA?
a) 0.1 - 3 kb
b) 0.5 - 7 kb
c) 1 - 20 kb
d) 5 - 60 kb
a) 0.1 - 3 kb
Why do scientists load DNA of known sizes (DNA ladder into the agarose gel?
It makes it easier to determine sizes of unknowns using comparison techniques.
What might the phrase run to red mean?
Your DNA fragments are moving towards the anode.
DNA will migrate through the gel towards the positive electrode since DNA has a strong negative charge.
How can you tell if the gel electrophoresis is running properly?
There are bubbles at the negative electrode.
What does the agarose gel electrophoresis apparatus consist of?
- power pack
- gel tray
- gel comb
- buffer tank
What cannot be a reason for using agarose gel electrophoresis?
a) comparing two sets of DNA
b) organising DNA by shape of backbone
c) organising DNA fragments from largest to smallest
d) organising DNA in order to see fragment sizes
b) organising DNA by shape of backbone
Electrophoresis cannot arrange molecules on shape of backbone.
Why do smaller fragments migrate faster in the agarose gel?
They can easily fit the agarose matrix pores, and thus they can move through the gel quicker.
Why is it necessary to purify the PCR fragment?
It is important to separate it from the PCR reaction buffer and obtain the fragment at a higher concentration.
You PCR fragment has concentration of 55 ug/ul, how do you create a 15 ul sample at 15 ug/ul?
(mention the answer as how much ul you add of the urified fragment, and how much ul of sterile water)
You take 4.1 ul of purified fragment, and add it to 10.9 ul of sterile water.
(15 ul x 15 ug/ul)/55 ug/ul = 4.1 ul of purified fragment
15 - 4.1 = 10.9 ul of sterile water
