Tai lieu thuc hanh danh cho lop Thu Y tien tien

Different pipettes will measure a different range of volumes; this is written to the top of the button on the top of the pipette or on the body of pipette. Do NOT wind the pipette outs[r]

Group ID:……… Semester:………Year:………

Name:……… Student ID:………

Class:………

Name:……… Student ID:………

Class:………

TABLE OF CONTENTS Practice 1 DNA extraction……….1

Task 1: Learning to use a pipette………1

Task 2: Extraction of DNA from swine blood……… 6

Practice 2 Polymerase Chain Reaction (PCR)………9

Task 1: Dilute your genomic DNA sample……… 10

Task 2: Swine PCR………10

Practice 3 Electrophoresis……… 13

Task 1: Agarose electrophoresis……… 13

Task 2: Analyse result from swine PCR………15

General Practice……….18

Practice 1:DNA EXTRACTION

Task 1: Learning to use a pipette

Pipettes are instruments used to accurately measure and transfer small volumes of liquids Misusewill lead to inaccurate measurements and damage to the pipette In this task, you will learn to use a piston air displacement pipette with disposable tips

A Parts of a micropipette

a A curved piece for catching hold pipette

b. Close the pipette with your fingers

c Digital volume indicator

d Tip ejector button

Before coming to class

You will need to bring a laboratory coat to this class, wear glove when conducting experiment and tie back long hair

e

f

e Plunger buttonand Volume adjustment dial

f Attachment point for a disposable tip

B Sizes of micropipettes

Different pipettes will measure a different range of volumes; this is written to the top of the button on the top of the pipette or on the body of pipette Do NOT wind the pipette outside this range, as in this will result in a loss of calibration

The dial on the pipette indicates of volume of liquid that will be transferred The dial can have decimal places indicated by number in red The small strokes between each bottom number are used to dial values between the printed numbers

Here are the pipettes that we will use:

Pipette Dial numbers Comments

P10 00.0 to 10.0

(0 µl to 10.0 µl)

Decimal numbers in red Do Not wind below zero or above 10.0

P20 02.0 to 20.0

(2 µl to 20.0 µl)

Decimal numbers in red Do NOT wind below

2 or above 20.0 P40 05.0 to 40.0

(5 µl to 40.0 µl)

Decimal numbers in red Do NOT wind below

5 or above 40.0

P100 010 to 100

(10 µl to 100 µl)

No decimal numbers Do NOT wind below 10

or above 100.0

P200 020 to 200

(20 µl to 200 µl)

No decimal numbers Do NOT wind below 20

or above 200.0

P1000

010 to 100

(0.1 mL to 1.00 mL = 100µl to 100 µl)

Red number indicates number before decimal if expressed in mL (not µL) Do NOT

wind below 100 or above 1000µl (100)

Different tips fit different pipettes and measure different volumes:

C Adjusting Volume on micropipettes and inserting the Tip

The tips are racked in plastic boxes with covers When you receive a box, it will be sterile Please be careful when touching box or tips not to contaminate them The box should be closed when not in use to prevent airborn contamination

Inserting the Tip:

1 Select the correct size tips

2 Open the box without touching the tips with your hands

3 Insert the micropipette shaft into the tip and press down firmly This will attach the tip to the shaft

4 Remove the micropipettor with the tip attached

5 Close the box without touching the tips with your hands

Clear tips 0-10 µl Yellow tips 10-200 µl Blue tips 200-1000 µl

The plunger button (thumb knob) allows you to adjust the volume that is measured

It can be dialed to the left or right to increase or decrease the volume

The digital readout shows the volume that will be measured As you turn the volume adjustment dial, the numbers in the digital readout will change

Figure.A-C, Three positions of the button of the pipette D-H, Liquid handling with

pipettes D, Adjusting the volume E, Fastening an appropriate tip F, Drawing of the liquid G, Transferring the liquid into an Eppendorf tube H, Dispensing the liquid

1.Which pipette will you use to measure the following volumes? What will the dial read when set to measure the required volume? Which color tip will you use?

Amount to

Measure Which Pipette? (name) Dial Reading

Which colour tip?

2.3 µl

154.6 µl

783 µl

36.2 µl

19.0 µl

D Practice:

Within your groups of 4 students, work in pairs to use pipette You will have 3 Petri dishes each containing a piece of filter paper

a) Label the filter papers as follows:

Petrie dish 1 : 5 µlPetrie dish 2: 90 µlPetrie dish 3: 250 µl

b) Each pair should measure the given volume of coloured liquid into one side of the Petri dish

How to do?

1 Select the correct pipette and the dial required volume If the dial doesn’t move, push the locking bar down

2 Open the container containing the liquid to be aspirated and the container where the liquid will be dispensed

3 Select the correct tip size

4 Hold the tip box steady with one hand Holding the pipette in your dominant hand and keeping the pipette vertically, place the pipette into the tip and twist about 180 degrees to add the tip Raise out of the box Be careful to keep the tip clean is not to touch anything with the tip

5 Press and HOLD the top button (piston) down to the FIRST stop

6 Keeping the pipette vertical, lower the tip just below the surface of the liquid to

be

7 Slowly raise the top button until you can let go of the button

8 Raise the pipette tip out of the liquid

9 Place the tip into the container where the liquid is to be dispensed Commonly, the tip is place against the side of the container

10 Gently press the top button (piston) down to the FIRST stop, then continue pressing to the SECOND stop (which blows out the small amount of liquid left in the tip) and HOLD down the button while you lift the pipette out f the container

11 Slowly release the button Use the tip eject button (coloured) to expel the tip into the waste container

Are all the dye circles in the Petri dish the same size?

Task 2: Extraction of DNA from swine blood

A Purification of swine blood sample

Eliminating all DNA – free components (plasma, serum, hemoglobin, platelet in mammal blood) This step critically contributes to the purity of DNA after the purification process

is complete

Note: 2 students per blood sample

Clean up after class (including all used test tubes)

Tools: Centrifuge, 15ml test tubes for containing samples, micropipette, pipette filler

bulb, jar for containing eliminated liquid

Chemical: NaCl 0.2%

Procedure:

1 Draw 5ml of NaCl 0.2% into test tube containg 1ml swine blood, mix thoroughly and centrifuge at 3000rpm for 3 minutes

2 Remove the supernatant

3 Repeat (1) and (2) 4 times

B Extract of DNA from swine blood

The isolation of genomic DNA is the first step of many genetic protocols used in genetic disease testing, paternity testing, forensics and in research The methods we use today are among the methods currently used in resaerch and testing laboratories You will gain experience in extracting DNA from swine blood

Note: 2 students per blood sample

Clean up after class (including all used test tubes)

Tools: Centrifuge, micropipette, tips, jar for containing eliminated liquid

Chemical: Lysis Buffer, Proteinase K, Phenol, CIAA (Chloroform/Isoamyl Alcohol),

Ethanol 95%, Ethanol 70%, TE 1X

Procedure:

1 Add 3ml Lysis buffer and 30 µl Proteinase K to the sample-containing test tube, mix thoroughly and incubate at 55oC over night (at least 8 hours)

Q2.1 What is the role of the Proteinase K enzyme?

2 Add 3ml Phenol, mix and centrifuge at 3000rpm for 5 minutes Ensure that the centrifuge is balanced before starting

Q2.2 Why are you centrifuging your sample?

Q2.3 Precipitation should appear in this step Why can it be seen now?

Q2.4 Where is the DNA now?

3 Transfer the supernantant into anew tube and add 3ml Phenol/CIAA, mix and centrifuge at 3000rpm for 5 minutes Ensure that the centrifuge is balanced before starting

Q2.5 Where is the DNA now?

4 Transfer the supernatant into a new tube and add 3ml CIAA

(Chloroform/Isoamyl Alcohol), mix and centrifuge at 3000rpm for 5 minutes

Q2.6 Where is the DNA now?

5 Transfer the supernatant into a test tube containing 5ml ice-cold Ethanol and invert gently to mix

Q2.7 White strand should appear What is this and why can it be seen now?

6 Centrifuge at 6000rpm for 5 minute You should see a pellet near the base of the tube Remove the supernatant, ensuring the pellet is not discarded

7 Add 3ml Ethanol 70%, invert 50 times to wash the pellet and centrifuge at

3000rpm for 5 minutes

8 Remove all ethanol solution and let the sample dry

9 Add 200 µl TE 1X buffer to dissolve the pellet Gentle flicking of the tube will encourage the pellet to dissolve

10 Incubate at 370C over night

11 Transfer the DNA solution into eppendorf 1.5ml.Ensure your tube is labelled with your assigned number

Practice 2: POLYMERASE CHAIN REACTION (PCR)

The purpose of this practical is to give you some experience in a commonly used approach for many making many copies of a specific region of the genome through in

vitro DNA replication using the polymerase chain reaction or PCR PCR is the starting

point for many analytical techniques, including

 microsatellite genotyping to identify individuals

 the sequencing of genes to analyze variation associated with genetic diseases

 the diagnosis of certain infectious diseases

 molecular sexing where species do not show external, or early, indicators of sex (e.g birds) or where mature animals cannot be examined (e.g embryos or faeces/hair samples in conservation genetics)

In this way, PCR forms an integral part of most genetic tests for disease and disease causing agents

PCR is able to amplify even extremely small amount of DNA for analysis This means that

it can be used to amplify DNA extracted from forensic samples like hair, traces of blood

or even single sperm However, this also mean that very small amount of contaminating DNA will also be amplified You should show extreme caution in ensuring that only DNA from your target sample will be in your PCR reaction!

Within your group of 4 students, works in pair to set up the swine PCR Each pair will set up a PCR for the 4 DNA samples (1 from your own group and 3 from other group)

You will be allocated a group number Record your group number here: _

So the group of 4 students will label in group 3 will have tubes labeled: 3-1,3-2

Before coming to class

You will need to bring a laboratory coat to this class, wear glove when conducting experiment and tie back long hair

Review the principles of PCR

Task 1:Dilute your Genomic DNA sample

1 For each sample, you will need to dilute the DNA Do this for your own swine DNA sample

a record the concentration of your DNA sample in column C This value will be provided to you by your tutor

b Calculate how much DNA and water you need to add to prepare 200 µl of DNA diluted to 10 ng/µl using the following table

i You can use the formula initial concentration X initial volume = final concentration X final volume or column D = (A x B)/C

ii How much water will you need to add to bring the total volume to 200 µl? i.e column E = B – D

Sample

ID

Final

concentration

Final volume Current

concentration

(“initial concentration”)

Amount of DNA

to add

(“initial concentration”)

Amount of water to add

10 ng/µl 200 µl

2 Use a new 1.5 mL eppendorf tube and add the amount of water from column E and the amount of DNA from column D

Task 2: Swine PCR

1 To set up the PCR, you will need to make a Master Mix for your group, with one

reaction for each DNA (one from your own sample and the other three from other

group), one reaction for a negative (no DNA template) control for PCR

Q2.1 Why is a negative for the PCR also needed?

2 You will need a 200µl eppendorf tube per group (2 students) to make up the master mix Label this tube with your group number

3 Calculate how much of each reagent you will need to add to your master mix using the table shown below

a Column A shows you initial concentrattion Column B shows you

concentration for running PCR Column C shows shows you the amount needed for 1 reaction And column D shows the amount needed for 4 reaction plus 1 (negative)

b To complete column C, calculate by these equations :

- “Rule of three”

- C = B x V∑/A

- H2O = V∑ - known reagents

Sterile pure H2O

Forward primer 10pmol/µl 50pmol

Reverse primer 10pmol/µl 50pmol

Enzyme (Taq DNA

polymerase)*

4 Add each reagent to your master mix tube Mix by gently flicking or vortexing Keep on ice

5 Label a 0.2ml (small eppendorf) tube for each sample – label with group number and pair of group Eg for group 2, pair 1: label 2-1 Note you should label on the side, not on the top (labels on the top will removeed by hot lid of the PCR

machine) Negative tube, label N

Record the details of the samples here:

1

2

3

4

5

6 Pipette out 15µl of the master mix into each of the small reaction tube

7 Add 5µl of DNA of the relevent DNA into each tube following the list in step 2 above Do not add anything to the N tube

8 Give tubes to the tutor Samples will be run on together on a PCR machine using the programme:

INITIAL DENATURATION 940C, 10 min

Then

DENATURATION 940C, 30 sec

ANNEALING 550C, 45 sec Repeat this for 35 cycles

EXTENSION 720C, 45 sec

Then

FINAL EXTENSION 720C, 5 min

HOLD 40C

Q2.2 How long (approximately) will the PCR take?

Practice 3: ELECTROPHORESIS

Task 1: Agarose electrophoresis

A Agarose 1% preparation (carried out by lecturer)

1 Weigh 1g agarose and 100ml TAE 1X solution

2 Microwave to dissolve the agarose (achieve boiling point 3 times)

3 Wait until the agarose cools down (500C) and add 6 µl ETBR Gently shake the agarose over ice clockwise, then counterclockwise to avoid bubbles and help ETBR dissolve completely in gel ETBR intercalates into double-strand of DNA and flouresces when placed under UV light

4 Pour the gel into the mold and let it fix

Q3.1 How many grams of agarose are needed to prepare 100ml agarose solution of 1.5%?

Q3.2 Why should bubbles be avoided when the gel is prepared?

B Sample loading (carried out by students)

1 When the groups located to a gel are ready, add the gel to electrophoresis tank Remember the DNA will migrate from black to red (negative to positive) so sit your gel in the correct way

2 Observe the tutor loads 5µl ladder 100 (ABM) into the wellof an agarose gel

Before coming to class

Note: Today’s class involves the use of EtBr, a DNA stain, which may be mutagenic and is carcinogenic All students should handle the gels and buffers with nitrile gloves and follow the instruction of the tutors