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]
(1)1 Group ID:……… Semester:………Year:………
Name:……… Student ID:……… Class:………
Name:……… Student ID:……… Class:………
TABLE OF CONTENTS
Practice DNA extraction……….1
Task 1: Learning to use a pipette………1
Task 2: Extraction of DNA from swine blood………
Practice Polymerase Chain Reaction (PCR)………9
Task 1: Dilute your genomic DNA sample……… 10
Task 2: Swine PCR………10
Practice Electrophoresis……… 13
Task 1: Agarose electrophoresis……… 13
Task 2: Analyse result from swine PCR………15
(2)2
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
(3)3 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 00.0to 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 or above 20.0
P40 05.0 to 40.0 (5 µl to 40.0 µl)
Decimal numbers in red Do NOT wind below 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)
(4)4 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
(5)5
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 students, work in pairs to use pipette You will have Petri dishes each containing a piece of filter paper
(6)6 Petrie dish : 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 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
(7)7
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: 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 minutes
2 Remove the supernatant Repeat (1) and (2) 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: students per blood sample
Clean up after class (including all used test tubes)
Tools: Centrifuge, micropipette, tips, jar for containing eliminated liquid
(8)8
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 hours)
Q2.1 What is the role of the Proteinase K enzyme?
2 Add 3ml Phenol, mix and centrifuge at 3000rpm for 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 minutes Ensure that the centrifuge is balanced before starting
(9)9 Transfer the supernatant into a new tube and add 3ml CIAA
(Chloroform/Isoamyl Alcohol), mix and centrifuge at 3000rpm for 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 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 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
(10)10
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 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 students, works in pair to set up the swine PCR Each pair will set up a PCR for the DNA samples (1 from your own group and from other group) You will be allocated a group number Record your group number here: _
So the group of students will label in group 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
(11)11
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
A B C D E
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
(12)12 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 reaction And column D shows the amount needed for reaction plus (negative)
b To complete column C, calculate by these equations : - “Rule of three”
- C = B x V∑/A
- H2O = V∑ - known reagents
Reagents A B C D
DNA 10ng/ µl 50ng
Sterile pure H2O
PCR buffer 10X 1X
dNTPs 1.0mM 150µM
Forward primer 10pmol/µl 50pmol Reverse primer 10pmol/µl 50pmol
MgCl2 25mM 2,25mM
Enzyme (Taq DNA polymerase)*
5UI/ µl 1UI
Total (V∑) 20 µl
4 Add each reagent to your master mix tube Mix by gently flicking or vortexing Keep on ice
(13)13 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 Add 5µl of DNA of the relevent DNA into each tube following the list in step
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 Then
DENATURATION 940C, 30 sec
ANNEALING 550C, 45 sec Repeat this for 35 cycles EXTENSION 720C, 45 sec
Then
FINAL EXTENSION 720C, HOLD 40C
(14)14
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 times)
3 Wait until the agarose cools down (500C) and add 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
(15)15 Each student carefully pipette 5µl of PCR product into an agarose gel Note that
each sample should be loaded and electrophoresis started without delay Write here the your sample and order samples on gel
4 Ensure the power pack is switched off, then place the lid in place Connect the electrodes from the electrophoresis tank to the power pack (red to positive, black to negative)
5 Set the voltage to 100 volts and press Start Run the eletrophoresis for 30 minutes
6 When the electrophoresis run is complete, the tutor will turn the power pack off and remove the gel, place your gel under UV light
7 Observe and determinate your result
Q3.3 What result did you obtain from your PCR?
Q3.4 The size of PCR products:………bp
Q3.5 Your sample is (positive/negative)……… for (PRRS/PED) ………
(Note: to answer Q3.5, answer the questions in Task first)
Q3.6 If one or more bands are seen in the ‘negative’ lane, what does it mean?
(16)16 Q3.8 Fill in the sizes corresponding to bands of ladder
Figure Electrophoresis with ladder 100
Task 2: Analyse results from swine blood
Each pair will obtain a result from your PCR which is expected to diagnosis of certain infectious disease: PRRS or PED
Q3.9 PRRS is the shorthand abbreviation of……… ……… Also known as……… … that occurs in……… ……….……… , caused by the ……… virus
Q3.10 PED is the shorthand abbreviation of: ……… ………….……… caused by……… ………virus
(17)17 F is………
R is………
Q3.11 Find the primer sequences in the sequences shown below Underline the primer sequences in both the sequences
(Hint: you need to write the reverse complement sequence of the reverse primer)
LOCUS AY612613
14281 GCAAAGTTGA GGTCGAAGGT CATCTGATCG ACCTCAAAAG AGTTGTGCTT GATGGTTCCG 14341 TGGCAACCCC TATAACCAGA GTTTCAGCGG AACAATGGGG TCGTCCTTAG ATGACTTCTG 14401 TCATGATAGC ACGGCTCCAC AAAAGGTGCT TTTGGCGTTT TCTATTACCT ACACGCCAGT 14461 GATGATATAT GCCCTAAAGG TGAGTCGCGG CCGACTGCTA GGGCTTCTGC ACCTTTTGAT 14521 CTTCCTGAAT TGTGCTTTCA CCTTCGGGTA CATGACTTTC GCGCACTTTC AGAGTACAAA 14581 TAAGGTCGCG CTCACTATGG GAGCAGTAGT TGCACTCCTT TGGGGGGTGT ACTCAGCCAT 14641 AGAAACCTGG AAATTCATCA CCTCCAGATG CCGTTTGTGC TTGCTAGGCC GCAAGTACAT 14701 TCTGGCCCCT GCCCACCACG TTGAAAGTGC CGCAGGCTTT CATCCGATTG CGGCAAATGA 14761 TAACCACGCA TTTGTCGTCC GGCGTCCCGG CTCCACTACG GTCAACGGCA CATTGGTGCC 14821 CGGGTTAAAA AGCCTCGTGT TGGGTGGCAG AAAAGCTGTT AAACAGGGAG TGGTAAACCT 14881 TGTCAAATAT GCCAAATAAC AACGGCAAGC AGCAGAAGAG AAAGAAGGGG GATGGCCAGC 14941 CAGTCAATCA GCTGTGCCAG ATGCTGGGTA AGATCATCGC TCAGCAAAAC CAGTCCAGAG Primer Sequences of the primers (5’ – 3’) Infection
detected
Amplified target
gene
Size of PCR product
ORF6-F GTGGCAACCCCTATAACCAGAG
PRRSv ORF6 780 bp ORF6-R ACGACAGACACAATTGCCGCTCAC
E pro-F CGCAGTTTACACACCTATAGGG
PEDv E&M 412 bp
(18)18
15001 GCAAGGGACC GGGAAAGAAA AATAAGAAGA AAAACCCGGA GAAGCCCCAT TTTCCTCTAG 15061 CGACTGAAGA TGATGTCAGA CATCACTTTA CCCCTAGTGA GCGGCAATTG TGTCTGTCGT 15121 CAATCCAGAC CGCCTTTAAT
//
F:………
R:………
Size of PCR product:………
LOCUS JQ282909
25561 GCTTCACTTG TCACCGGTTG TGTAATAGCG CAGTTTACAC ACCTATAGGG CGTTTGTATA 25621 GAGTTTATAA GTCTTACATG CAAATAGACC CCCTCCCTAG TACTGTTATT GACGTATAAA 25681 CGAAATATGT CTAACGGTTC TATTCCCGTT GATGAGGTGA TTCAACACCT TAGAAACTGG 25741 AATTTCACAT GGAATATCAT ACTGACGATA CTACTTGTAG TGCTTCAGTA TGGCCATTAC 25801 AAGTACTCTG CGTTCTTGTA TGGTGTCAAG ATGGCTATTC TATGGATACT TTGGCCTCTT 25861 GTGTTAGCAC TGTCACTTTT TGATGCATGG GCTAGCTTTC AGGTCAATTG GGTCTTTTTT 25921 GCTTTCAGCA TCCTTATGGC TTGCATCACT CTTATGCTGT GGATAATGTA CTTTGTCAAT 25981 AGCATTCGGT TGTGGCGCAG GACACATTCT TGGTGGTCTT
//
F:………
R:………
(19)19
General Practice
Based on the paper, answer and complete the information (by Vietnamese): The title of paper, name of authors?
2 Purposes and objects?
(20)20 Complete the tables below:
Reagent Amount needed for reaction
Running
programme Temperature Time
DNA Initial
denaturation Sterile pure H2O Denaturation
PCR buffer Annealing
dNTPs Extension
Forward primer Final extension
Reverse primer Number of cycle
MgCl2
Enzyme (Taq DNA
polymerase)* Total (V∑)
5 How long (approximately) will the PCR take?