Lab 2

Question 1

What happens in lab 2?

Answer 1

• Completion of genomic DNA quantification
• Control experiment: Examination of isolated genomic DNA by agarose gel electrophoresis
• Control experiment: PCR amplification of a specific, known genomic DNA fragment

Question 2

What is the control experiment and what will it tell us? 3

Answer 2

1 * Control experiment: Examination of isolated genomic DNA by agarose gel electrophoresis.

• This experiment will show us if

2. . the DNA is degraded or of a high molecular weight
3. . if the DNA is contaminated with RNA

Question 3

Control Experiment: Examination of Isolated
Genomic DNA by agarose gel electrophoresis

Answer 3

• You will examine the quality of the isolated genomic DNA from Session 1 by 0.6% (w/v) agarose gel
  electrophoresis

Question 4

Why is the examination of Isolated Genomic DNA by
agarose gel electrophoresis important?

‘Question: Is the isolated genomic DNA of high enough quality for an AFLP experiment?’ = 2

Answer 4

• AFLP experiments rely on restriction digests generating fragments of defined
  SIZES
• If the DNA is partially degraded before the digest, the fragments obtained will NOT REPRESENT TRUE SIZES OF RESTRICTION FRAGMENTS

Question 5

Why is the examination of Isolated Genomic DNA by
agarose gel electrophoresis important? … diagram

Answer 5

AFLP with intact gDNA

vs

AFLP with partially degraded gDNA

slide 6

Question 6

Analysing DNA using agarose gel electrophoresis…GOOD GENOMIC DNA IS = 2

Answer 6

Good genomic DNA
* High molecular weight band = intact gDNA

• No bands in lower part of gel = RNA free
  …NO BANDS IN LOWER ‘MW’ = FREE OF RNA

Question 7

Analysing DNA using agarose gel electrophoresis..

Degraded/low quality genomic DNA = 2

Answer 7

Degraded/low quality genomic DNA

• Medium to low molecular weight band
  smear = degraded gDNA
• Bands in lower part of gel = RNA

Question 8

Which agarose concentration should you use for your DNA analysis? = 4

Answer 8

1 * This will depend on the size of the DNA fragments
you want to separate ➔ see table. (SLIDE 9)

2 * It also depends a bit on the running buffer used.

3. • Usually, you get better separation if the gel is run
     at a lower voltage, but it will take more time. The DNA bands will look sharper if the gel is run at lower voltage.
4. • Running the gel at lower temperatures also
     improves the sharpness of the bands as less
     diffusion of the DNA occurs.

Question 9

FAQ: How do you know how much agarose solution you
need

Answer 9

1 * Remember 1 mL = 1 cm3

2 * Measure the width and length of the inside of
the gel tray. Example 7 cm wide and 8 cm long.

3 * The product of the two values is 56 cm2

4 * If your gel should be 1 cm high (quite a thick gel), you would need 56 mL of agarose solution

– Why?
* 56 cm2 x 1 cm = 56 cm3 = 56 mL

Question 10

What is GelRed? = 7

Answer 10

1 * GelRed consists of two ethidium subunits that are connected by a
spacer.

2 * It intercalates into DNA = it inserts between the planar bases of DNA.

3 * GelRed is a fluorophore. When exposed to UV light, GelRed will Fluoresce with an orange colour. The fluorescence of GelRed is much stronger when bound to DNA

4 * The longer the DNA fragment or the more DNA is present, the stronger is the fluorescent signal. WHY? More GelRed can intercalate into longer DNA molecules and into more DNA molecules.

5 * It is very sensitive and allows for detection of very small amounts of DNA in an agarose gel

6 * It is safer to use than ethidium bromide

7 * It is stable at room temperature

Question 11

What is the role of TBE buffer? = 5

Answer 11

1. Tris – Borate – EDTA (pH 8.0)
2. • Ions conduct electricity
3. • Tris-acid solutions: buffers for slightly basic conditions keeps DNA deprotonated and soluble in water.
4. • EDTA: chelator of divalent cations, particularly of magnesium (Mg2+) which is a necessary co-factors for nucleases ➔ protect the nucleic acids against enzymatic degradation
5. • Borate: inhibits enzymes ➔ protects DNA from degradation

Question 12

What is the role of DNA loading dye? = 4

Answer 12

1. Bromophenol blue Gel loading buffer
2. • Glycerol: makes samples denser than the running buffer so that the samples sink in the well.
3. • Bromophenol blue: dye to assess how “fast” your gel is running. Usually runs at
     a lower molecular weight than most DNA molecules.
4. • EDTA: chelator of divalent cations, particularly of magnesium (Mg2+) which
     is a necessary co-factors for nucleases ➔ protect the nucleic acids against enzymatic degradation.

Question 13

How to load a sample into an agarose gel?

Answer 13

1. Ensure that you move the pipette tip to just above the loading wells in the gel
2. If your sample moves out of the pocket it may contain ethanol from the DNA preparation. Make a new sample adding twice as much loading dye.

Question 14

In which direction does DNA migrate in an agarose gel?

Answer 14

DNA is negatively charged in TBE buffer.

Hence it migrates from the cathode (negative) to the anode (positive).

Ensure the loading wells of the gel are on the side of the cathode.

Remember
* The negative anion moves to the positive anode.
* The positive cation moves to the negative cathode.

Question 15

What is a 10 x solution? = 4

Answer 15

1. • A 10 x solution is a stock solution that is 10 fold higher concentrated then it is used in a reaction. The amount of reagent required in a reaction is by definition 1 x.

2 * To get from a 10 x stock solution to a 1 x concentration in a reaction, one has to dilute the 10 x stock solution 1:10.

3. • Similarly a 20 x stock solution has to be diluted 1:20.
4. • Example:
     ▪ A loading dye is 20 x concentrated and used as 1 x in the loaded sample .
     ▪ The total volume of the sample to be loaded should be 10 μL.
     ▪ Dilute the loading dye 1:20 in the sample volume of 10 μL ➔ add 0.5 μL of the loading dye. Why? Because 0.5 μL of a
     20 x stock solution made up to a total volume of 10 μL is a 1:20 dilution that results in a final concentration in the
     reaction of 1 x. You can check this with the following calculation:
     ▪
     0.5 μL ∗20 x
     10 μL
     = 1 �

Question 16

GENE3370: AFLP Lab 2 Program

• Control experiment: PCR amplification of a specific, known genomic DNA fragment

Answer 16

• ‘Control experiment: PCR amplification of a specific, known genomic DNA fragment.’

➢ This experiment will show us if the DNA is free of contaminants that can inhibit a PCR reaction.

➢ Expect one PCR product of known size (session 3)

Question 17

What goes into a PCR reaction and why?

WATER

Answer 17

Ensures that the concentrations of all products is correct

Question 18

What goes into a PCR reaction and why?

TAQ POLYMERASE

Answer 18

This enzyme is heat stable, reads template DNA sequence, selects
complementary dNTP and adds it to the primer/growing DNA strand

Question 19

What goes into a PCR reaction and why?

MAGNESIUM

Answer 19

Cofactor for Taq polymerase, stabilises DNA hybrids and
Taq:DNA/dNTP interactions

Question 20

What goes into a PCR reaction and why?

dNTP

Answer 20

Are the building blocks for the new DNA, used by Taq polymerase

Question 21

What goes into a PCR reaction and why?

PCR BUFFER

Answer 21

Keeps pH constant, often supplies additional ions required for a
PCR (KCl, (NH4 )SO4 etc)

Question 22

What goes into a PCR reaction and why?

TEMPLATE DNA

Answer 22

Sequence serves as template/information for the synthesis of new DNA strands

Question 23

What goes into a PCR reaction and why?

REVERSE PRIMER

Answer 23

Anneals/hybridises to forward DNA strand and acts as starting point for reverse strand synthesis

Question 24

What goes into a PCR reaction and why?

FORWARD PRIMER

Answer 24

Anneals/hybridises to reverse DNA strand and acts as starting point for forward strand synthesis

Question 25

What goes into a PCR reaction and why? = 8

Answer 25

1. Forward primer
2. reverse primer
3. template DNA
4. PCR buffer
5. dNTP
6. Magnesium
7. Taq polymerase
8. Water

Question 26

Control amplification of a specific DNA fragment = 4

Answer 26

• You will prepare two PCR reactions

1) Contains genomic DNA as template. If the genomic DNA is of good quality, you will obtain a PCR
product.

2) Contains plasmid DNA. We have cloned the genomic DNA fragment you will amplify under 1) into a
plasmid. This PCR serves as a positive control for the PCR reaction, it should result in a PCR fragment if you set-up the reaction correctly.

3… * Each PCR reaction contains the same components except for the DNA which will be added later to start the reaction

4…* You will all of the common components from a mastermix into a PCR tube and then add the DNA

Question 27

Calculating dilutions

Answer 27

• A stock solution of 10 pmoles/μL should be used to achieve a concentration of 1 pmoles/μL in a reaction. This requires a
  1:10 dilution of the stock solution.
• Example:
• A primer stock solution has a concentration of 10 pmoles/μL
• The final volume in the PCR reaction should be 25 μL.
• Dilute the primer stock solution 1:10 in the reaction volume by adding 2.5 μL of the primer stock solution into a total
  volume of 25 μL.
• You can check this by calculating:
• 2.5 μL ∗ 10 pmoles/μL
  25 μL
  = 1 pmoles/μL
• You proceed in a similar way for a stock solution that is 10 mM with a required concentration in the reaction of 1 mM.

Again, this requires a 1:10 dilution. You will add 2.5 μL of the stock solution into a total volume of 25 μL.

Question 28

What does U/ μL mean and how do I use it? = 7

Answer 28

1 * The U in U/ μL is used for enzymes. The U stands for units of activity.

2 * The unit tells us something about the activity of the enzyme.

3 * For example, it could mean that 1 unit (U) is the amount of enzyme that catalyses a reaction of 1 μmol of substrate per minute

4 * An enzyme with 5 U/ μL has 5 enzymatic units in one μL .

5 * If we need 1 unit of the enzyme per reaction, we need to add 1:5 of one microliter of the enzyme to the reaction volume.

6 * Example:
▪ An enzyme has a ‘concentration’ of 5 U/ μL. One unit of the enzyme should be used in 30 μL of the reaction.

One unit is in 0.2 μL of the concentrated enzyme. Thus, we have to add 0.2 μL of the concentrated enzyme (5
U/ μL) to the reaction. NOTE: this is independent of the reaction volume.

Question 29

Mastermix: 6

Answer 29

1 * A mastermix is a very time saving concept that
contributes greatly to accuracy of an
experiment.

2 * A mastermix contains all of the components
shared by several reaction.

3 * The mastermix is then added to each reaction
tube.

4 * The unique components are then added
individually to individual reaction tube.

5 * Make always more mastermix than you need to
account for pipetting errors. Usually for up to 10 reactions needed, one more is added. Thus, for five PCR reactions, one would make a mastermix for 6 reactions = five required reactions plus one spare

6.* In the PCR you will perform in session 2, primers, dNTPs, magnesium, Taq DNA polymerase and water are the common components that go into the mastermix. The DNA templates differ between the reactions and are the unique components.

Question 30

Calculating a Mastermix –

Answer 30

• Start the calculations for the mastermix by
  determining the volumes of the components for one
  reaction.
• To do this, you need to calculate the dilutions you
  have to make for each of the components using the
  concentration of the component and the final
  concentration (f.c.) of the components in one
  reaction.
• Example: you have a concentrated forward
  primer of 10 pmoles/μL and the f.c. should be 1
  pmoles/μL. This means you have to make a 1:10
  dilution of the primer.
• With a total volume in one reaction of 25 μL, a
  1: 10 dilution means you will have to add 2.5 μL
  primer into each reaction.
• The calculations for the other components are
  done in a similar way.
• Now, you have to calculate the amount of SDW to
  add.
• Each tube should have a final volume of 25 μL.
• With that and all the other volumes calculated on the
  previous slide, the volume of SDW for one reaction in
  the example is:
• VolumeSDW = volumetube - ∑volume components
• Volume SDW = 25 μL – 14.25 μL = 10.75 μL
• Now use the volumes for one reaction to calculate
  the volume for the mastermix (here 6 reactions).
• Prepare the mastermix by pipetting the volumes you
  calculated into a mastermix tube. Ensure you leave
  the DNA out as you will add a different DNA
  (=experimental variable) to the individual reaction
  tubes.
• You will add 1 μL DNA individually into
  individual reaction tubes.
• This leaves 24 μL for the mastermix to be
  added to each reaction tube giving a total
  volume of 25 μL.
• You can check this by adding all volumes of the
  mastermix and divide this by six reactions:
• Volume of mastermix = 144 μL
• Volume of mastermix/six reactions = 24 μ

Question 31

Control amplification of a specific DNA fragment (quality control)

Answer 31

• Add one 24 μL aliquot of the mastermix to each of two separate PCR tubes (small tubes)
• Add to one tube 1 μL genomic DNA. Add to the second tube 1 μL plasmid DNA (control template)
• Mix, microfuge briefly to collect sample add the bottom of the tube, place in thermocycler
• Amplification program
  94 ℃ 2 minutes
  94 ℃ 30 seconds - denature
  55 ℃ 30 seconds - anneal primers
  72 ℃ 45 seconds - extension
  72 ℃ 10 minutes
  4 ℃ hold
  35 cycles