Electrophoresis

Question 1

What is electrophoresis used for?

Answer 1

PCR products analysed by electrophoresis

DNA must be negatively charged

Question 2

Gel Electrophoresis

Answer 2

• nucleic acid electrophoresed through a gel
• gels have pores in them through which DNA is “sieved”
• large pieces of DNA are retarded, small pieces travel through gel more quickly

Question 3

Gels

Answer 3

• solid matrix with a series of pores and a buffer solution
• gel materials mixed with buffer and poured into a mould
• sample “wells” are created by placing a “comb” into the gel so that the teeth of the comb put wells into gel material.
• common gels = agarose, polyacrylamide

Question 4

What is agarose gel made from?

Answer 4

natural highly purified product from seaweed

Question 5

What is the diameter of pores in agarose gel?

Answer 5

200nm

Question 6

Agarose gel electrophoresis

Answer 6

• gels prepared by weighing out the required amount of agarose powder, adding it to buffer and melting mix in microwave
• mix is poured (after cooling to 55-60 degrees celsius) into a gel mould and the comb inserted so that the bottom of the teeth are just off the bottom of the gel.
• gel is allowed to set
• gel is submerged in buffer, samples mixed with loading dye and loaded in the gel
• agarose gels used at concentrations of 0.6% to 2%, although you can use more and specialised agaroses have been developed that can be used above 4%
• used to analyse relatively large pieces of DNA (500bp to 20,000 bp)

Question 7

Name 3 commonly used buffers.

Answer 7

TBE (tris-borate-EDTA)
TAE (tris-acetate-EDTA)
TPE (tris-phosphate-EDTA)

Question 8

Polyacrylamide gels

Answer 8

• formed chemically through polymerisation of acrylamide monomers and a cross linker
• free radicals from the decomposition of ammonium persulphate initiate polymerisation and TEMED stabilises the free radicals
• pore size can be altered by adjusting total acrylamide conc and the conc of the cross linker.

Question 9

Visualisation of DNA in gels

Answer 9

• common = ultraviolet
• ethidium bromide = stain DNA in gels
• ethidium bromide is an intercalating agent - chemical substance which has a structure like a flat plane so can insert itself in between adjacent nucleotides of DNA molecule
• when viewed under UV light the DNA fluoresces and becomes visible.

Question 10

What is now used instead of ethidium bromide to visualise DNA in gels?

Answer 10

SYBR green as safer

Question 11

Optimisation

Answer 11

components of PCR reaction and cycle parameters may need to be optimised:

• annealing temp of primers
• conc of Mg2+ in reaction
• extension time
• denaturing + annealing times
• extension temperature
• amount of template and polymerase

Question 12

Optimisation of annealing temp

Answer 12

primers have a calculated annealing temp
temp must be confirmed practically
temp steps of 2 degrees celsius above and below

Question 13

Optimisation of magnesium conc

Answer 13

vary Mg2+ in steps of 0.5M

sometimes a compromise between yield and specificity

Question 14

Choice of thermostable DNA polymerase

Answer 14

Taq DNA polymerase common but

• lacks 3’ to 5’ proof reading, usually found in DNA polymerases
• Taq makes a mistake with a frequency of one base in 104.
• mistakes occur randomly
• after 20 cycles, 33% of 400bp products may contain error
• can use proo reading enzymes e.g. pfu