Lab 1

Question 1

Define AFLP?

Answer 1

AFLP = Amplified Fragment Length Polymorphism

Question 2

WHAT IS THE AFLP:
Amplified Fragment Length Polymorphism METHOD?

Answer 2

1. A method to map gene locations and genotypes and to identify markers
2. Based on Restriction digest and PCR

Question 3

OUTLINE OF THE ‘AFLP’ PROCEDURE… 5

Answer 3

1. Restriction digestion
2. Adapter ligation
3. Preamplification (by PCR)
4. Selective Amplification (By PCR)
5. Gel electrophoresis

Question 4

Methods similar to AFLP: ‘RFLP’ ….UNDERSTANDING

Answer 4

• RFLP (Restriction Fragment Length Polymorphism) – based on restriction digest only

Question 5

Methods similar to AFLP: Understanding ‘RAPD’

Answer 5

RAPD (Random Amplification of Polymorphic DNA) – based on PCR only

• Both less suitable for larger genomes such as plant genomes. Produce less reliable results

Question 6

What is AFLP in more detail… 3

Answer 6

1. AFLP combines both, RE and PCR ➔ produces larger number of markers ➔ better chance of getting a useful marker that segregates with a phenotype
2. • Can be used to detect genetic differences between individuals within a population
3. • Useful in Biodiversity and epidemiology studies

Question 7

Understanding 2 pea varieties…

Generate AFLP patterns for 2 pea varieties..

Answer 7

1. CLIMBING PEAS
   - grow to a height of 2m and need trellising
   - tendrils are modified leaves and allow attatchment to the trellis
2. GRADEN PEAS
   - grow to a height of about 1m

Question 8

The two columns you will be using during the DNA preparation:

QIAshredder vs DNeasy mini spin columns

Answer 8

• The QIAshredder spin column is purple.
• The DNeasy mini spin column is colourless.

Question 9

AFLP lab 1: Isolation of DNA from Plant Tissue with the QIAgen DNeasy Plant Mini Kit

OVERVIEW OF THE STEPS = 17

Answer 9

1. Grind, lyse and precipitate
2. Centrifuge through QIAshredder spin column
3. Bind DNA to DNeasy mini spin column
4. Wash
5. Elute DNA
6. Percipitate unwanted components
7. and 8:
   Remove unwanted components by centrifugation through a QIAshredder column.

Step 9: Prepare
for binding to
DNeasy column

Steps 10 & 11:
bind DNA to
DNeasy column

Step 12 & 13:
Wash DNA

Step 14 to 16:
Elute DNA
-Washed DNA
bound to
DNeasy
column

-DNA is
ready to
use

Question 10

DNA extraction of pea variety you are working with (garden pea or climbing pea).

Answer 10

1.wearing safety glasses,

2. open the tube of plant tissue immediately upon receipt to prevent the lid from popping open and tissue being lost.
3. Label the tube of Buffer AE with your initials and preheat to 65 °C.
4. Check Buffer AP1 for precipitation. If a precipitate is present, heat briefly at 65,°C to re-dissolve, and mix well.

Question 11

Understanding steps 1- 5;

Releasing cell content: ‘Grind, lyse and perciptate’
= 8

Answer 11

1. This step has been done for you. Continue immediately with step 4.
2. • The tissue needs to be disrupted. This in done under LIQUID NITROGEN COOLING USING A MORTAR AND PESTLE.
3. receive the ‘powdered plant material’ in a tube which you OPEN TO ENSURE IT WILL NOT EXPLODE DUE TO NITROGEN PRESSURE.
4. • DISRUPTION of the tissue allows EFFICIENT EXTRACTION of DNA.
     COOLING ensures that the DNA isNOT DEGRADED
5. . You will add a CELL LYSIS BUFFER (AP1) and ‘RNase A’ TO the powdered plant
   TISSUE and MIX those well using a VORTEX MIXER.
6. • Note: Do not mix Buffer AP1 and RNase A prior to use.
7. • VORTEX well so that no tissue clumps are visible. Vortex or pipette
     further to remove any clumps. The cells in the CLUMPED TISSUE WILL NOT LYSE PROPERLY AND WILL THEREFORE RESULT IN A LOWER YEILD OF DNA.
8. . You will INCUBATE the mixture for ‘10 min’ at ‘65 °C.’
   - MIX 2–3 times DURING
   incubation by INVERTING THE TUBE = THIS STEP LYSES THE CELLS

Question 12

Removing unwanted components: Step 6 to 8

‘6. Percipitate unwanted components

7. and 8:
   Remove unwanted components by centrifugation through a QIAshredder column.’ = 7

Answer 12

1. You will add a BUFFER P3 to the LYSATE , mix by INVERTING THE TUBE, and
   incubate for 5 min ON ICE.
2. • This step PRECIPITATES detergent, proteins, and carbohydrates/
     polysaccharides (sugars) and lipids. THESE COMPOUNDS CAN INHIBIT DOWNSTREAM REACTIONS, INCLDUING THE ‘RE DIGEST AND PCR.’
3. You will use WIDE BORE pipette tips to apply the lysate to the purple
   QIAshredder spin column which you have placed in a 2 mL collection tube
   and centrifuge for 3 min at maximum speed.
4. The ‘QIAshredder’ REMOVES MOST PERCIPITATES AND CELL DEBRIS.. but a small
   amount may PASS THROUGH and form a PELLET in the collection tube. Be careful NOT DISTURB PELLET in step 8.
5. Wide bore pipette tips are used to AVOID SHEARING THE DNA

6.. You will TRANSFER FLOW THROUGH FRACTION from step 7 to a new 1.5 mL tube without disturbing the cell-debris pellet. ‘Typically, 450 μL of lysate is recovered.’

7. • For some plant species less lysate is recovered. In this case determine
     the volume using reverse pipetting to determine the lysate volume for
     the next step.

Question 13

DNA binding to DNeasy column: Step 9 to 12

‘Step 9: Prepare
for binding to
DNeasy column

Steps 10 & 11:
bind DNA to
DNeasy column’

= 8

Answer 13

1. You will add buffer AW1 to the QIAshredder eluate. You will need to add
   1.5 volumes of Buffer AW1 to the cleared lysate and mix by pipetting.
2. • Example: To 450 μL lysate add 675 μL Buffer AW1. Reduce the amount
     of Buffer AW1 accordingly if less lysate is recovered in step 8.
     For
     sample volumes that differ from 450 μL use the calculation:
     *
     X AW1 μL
     sample volume μL =
     675 μL
     450μL ➔ X (AW1) = 675 μL x sample volume μL
     450 μL
     (see in note book)
3. • A precipitate may form when adding AW1 ➔ this will not affect the DNeasy procedure.
4. • Note: pipette Buffer AW1 directly onto the cleared lysate and to mix
     immediately.
5. • AW1 contains ETHANOL and SALT and PREPARES the DNA FOR BINDING to the
     DNeasy column.
6. . You will apply 650 μL of the mixture from step 9, including any precipitate to the DNeasy mini spin column (this column is colourless) which you have placed into a 2 mL collection tube. You will centrifuge to
   remove the liquid and discard the flow through.
7. You will repeat step 10 if you had a greater volume 650 μL in setp 9
   than If you had more than 650 μL of the mixture from step 9.
8. Step 10 and 11 allow the DNA to bind to the DNeasy column

Question 14

DNA wash: Step 12 and 13

Step 12 & 13:
Wash DNA = 7

Answer 14

1. You will wash the DNA bound to the DNeasy column by adding BUFFER
   AW2 and CENTRIFUGATION.
2. • Discard the flow-through and reuse the collection tube in step 13.
3. • RemainingCONTAMINANTS such as protein and carbohydrates still present will be WASHED AWAY.

4.. You will repeat the wash by adding AW2 again and doing a longer centrifugation to ELUTE THE WASH BUFFER and to DRY THE MEMBRANE TO WHICH THE DNA IS BOUND.
the DNA is bound.

5. • At the end of the centrifugation, you have to ensure that you remove
     the DNeasy column from the collection tube carefully so the columnDOES NOT CONTACT THE FLOW-THROUGH AS THIS WILL RESULT IN CARRYOVER OF ETHANOL
6. • This SPIN ensures that NO RESIDUAL ENTHANOL is carried over during elution.
7. It is important to dry the membrane of the DNeasy column since
   residual ethanol may INTERFERE WITH SUBSEQUENT REACTIONS AND WITH LOADING OF SAMPLES INTO AN AGAROSE GEL

Question 15

DNA elution from DNeasy column: Step 14 to 17 = 7

‘Step 14 to 16:
Elute DNA
-Washed DNA
bound to
DNeasy
column

-DNA is
ready to
use’

Answer 15

1. You will transfer the DNeasy column to a new 1.5 mL microcentrifuge tube (with the lid removed) and pipette preheated (65 °C) Buffer AE directly onto the DNeasy membrane.
2. You will incubate the column for 5 min at room temperature and then
   centrifuge for 1 min at ≥6000 x g (≥9000 rpm) to elute the DNA.
3. You will repeat the elution steps 14 and 15 once with fresh Buffer AE,
   eluting into the same tube.

4 * The ‘buffer AE’ has no ethanol and low salt concentrations. Under those
conditions, the DNA is UNABLE TO BIND to the DNeasy column and will be
eluted by centrifugation.

5. Transfer the isolated DNA to a new 1.5 mL tube.
6. • Store the sample on ice to be used for setting up the DNA quantification.
7. Later, you will store the DNA at −20 °C until the next lab session

Question 16

Quantification of Genomic DNA using PicoGreen = 4

Answer 16

1. You will use the PicoGreen Double-Stranded DNA Quantitation Reagent to determine the concentration of DNA in your sample.

2.PicoGreen (Molecular
Probes), works similar
to SYBR Green (used in
qRT-PCR)

3. Both intercalate into
   DNA.
4. Once intercalated, the fluoresce strongly when irradiated with certain wavelengths of light

Question 17

Quantification of Genomic DNA using PicoGreen DIAGRAM

Answer 17

SLIDE 15

Question 18

Quantification of Genomic DNA using PicoGreen

You will use the PicoGreen Double-Stranded DNA Quantitation Reagent to determine the concentration of DNA in your sample. EXPLAIN…5

Answer 18

You will use the PicoGreen Double-Stranded DNA Quantitation Reagent to determine the
concentration of DNA in your sample.

1. Prepare two dilutions (1/200 and 1/400) of the DNA eluate in Tris-EDTA (TE) buffer. Prepare
   serial dilutions to decrease pipetting errors and MAKE AT LEAST ‘250 μL of each dilution.’ Mix and microfuge briefly to collect the contents at the bottom of the tube.
2. • TE buffer contains the buffer substance Tris which keeps the pH constant and EDTA (Ethylenediaminetetraacetic acid) which binds to and sequesters twofold positively charged ions.
3. It is used to complex Mg2+, a cofactor for enzymes that bind DNA. These enzymes include
   DNases that can break down DNA. Thus, EDTA PROTECTS DNA FROM ENZYMATIC DEGRADATION
4. • Store the diluted DNA in the rack provided. The technician will use this as shown in the next
     slide.
5. • Store the undiluted DNA at −20 °C until the next lab session.

Question 19

Quantification of Genomic DNA using PicoGreen –
steps done for you

Answer 19

1. This step will be done for you. Pipette 100 μL of each dilution into separate wells of a 96 well plate and
   add 100 μL of the PicoGreen reagent. Repeat in order to have duplicates. (Make sure that you note the
   location of each of your samples on the plate). A standard curve will be prepared for you using λ DNA and
   will cover the range 0 – 1000 ng/mL.

2.. The fluorescence intensity of the PicoGreen-DNA complexes will be measured and you will determine
the concentration of DNA in each sample using the equation of the line for the standard curve.

Question 20

What are the advantages of the PicoGreen method over the OD 260nm spectrophotometric method when measuring DNA concentrations = 5

Answer 20

1 ➢ ultra sensitive for quantitating double-stranded DNA

(dsDNA) in solution.
2 – Can detect as little as 250 pg/mL dsDNA

3 – Very low contribution of ssDNA and RNA to fluorescence

4 ➢ Wide dynamic detection range: assay is linear over four orders of magnitude with the same dye concentration

  5 – linearity is maintained in the presence of common contaminants: salts, ethanol, proteins etc.

Question 21

Standard curve format

Answer 21

X axis = wavelength (nm)

Y axis = fluorescence emission