Lab 1 Flow Chart : Learning basic laboratory skills
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Lab 1 Flow Chart : Learning basic laboratory skills RD Red dye solution S1 Dye 1 S2 Dye 2 S3 Dye 3 H 2 O Water 1 • Pick up and inspect the micropipettes on your bench, identify the parts. 2 • Transfer 50 μL water (H 2 O) into the 5 tubes. Avoid contamination: • Change a new tip. 2 3 4 5 1 • Label 5 microfuge tubes 1 through 5 using a marker. Plunger buon Tip ejector Display window Pipee p Barrel Which pipette to use? 3 Lab 1.1: Basic pipetting and serial dilution 50.0 µL H 2 O 1 2 3 4 5 4 50.0 µL RD 1 • Transfer 50 μL red dye (RD) into tube 1. • Pipette up and down several times to mix. 5&6 50.0 μL 1 2 3 4 50.0 μL 50.0 μL 50.0 μL • Use a new tip, transfer 50 μL solution from tube 1 into tube 2 and mix well. • Repeat the process for tube 2 through 5. • Observe the decreasing intensity of colour of redness from tube 1 through 5. 7 • Label a new microfuge tube gel RD. • Prepare 50-fold red dye solution (gel RD) by adding 196 μL water (H 2 O) , then 4 μL red dye (RD) to the tube. gel RD 196.0 µL H 2 O 5 & 4.0 µL RD gel RD 1X TAE buffer 1X TAE
Transcript of Lab 1 Flow Chart : Learning basic laboratory skills
Lab1FlowChart:LearningbasiclaboratoryskillsRD Reddyesolution
S1 Dye 1
S2 Dye2
S3 Dye3
H2O Water
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• Pickupandinspectthemicropipettes onyourbench,identifytheparts.
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• Transfer50 μLwater(H2O)intothe5tubes.
Avoidcontamination:• Changeanewtip.
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• Label5microfuge tubes1through 5usingamarker.
Plunger button
Tip ejector
Display window
Pipette tip
Barrel
Whichpipettetouse?3
Lab1.1:Basicpipettingandserialdilution
50.0 µL H2O
1 2 3 4 5
450.0 µL RD
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• Transfer50μL reddye(RD) intotube1.• Pipette upanddown severaltimestomix.
5&650.0 µL
1 2 3 4
50.0 µL 50.0 µL 50.0 µL
• Useanewtip, transfer50μL solution fromtube1into tube2andmixwell.
• Repeattheprocessfortube2through 5.• Observethedecreasingintensityofcolour of
rednessfromtube1through5.
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• Labelanewmicrofuge tubegelRD.• Prepare50-foldreddyesolution(gelRD) byadding
196 μL water (H2O) , then 4 μL reddye(RD) to the tube.
gelRD
196.0 µL H2O
5
& 4.0 µL RD
gelRD
1XTAEbuffer1X TAE
Lab1.2:Dyeseparationby gelelectrophoresis
9S1 S2 S3
• Centrifuge theS1,S2,andS3 tubes.
Balance theweight:• Arrangethetubesinatriangularpatterntoattain
uniformweightdistribution.
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• Draw the location of the wells in your notebook• Record whichsolution youwillplaceineachwell.
• Add10 μLS1solutionintothewell.• Repeat the step for S2 andS3.
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• Placethephotohoodonthegelelectrophoresis system• Turnonpowersupplyandpresstheon/offbutton.• Check ifbubbles form inthebufferatthe (-)electrode.
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• After10minutes,orwhenyoucandistinguish allthreedyes,turn offthe switch.
• Remove thephotohoodfromthe electrophoresissystem andobservethedyesinthegel.
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• Drawtherelativelocationofthebandsandtheir colorsineachofthelanescontaining yoursamples.
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••
Pour1xTAEbuffer(1xTAE) intothepractice plate. Practice yourtechnique bypipetting 10 µL 50-foldreddye solution (gelRD)toeachwell.
Makesurethewellsarenearthenegativeelectrode.
Well
Pipette tipS1
10.0 µL
TAE buffer
• Pour 1x TAEbuffer (1x TAE) into the gel tank.• Put the gel tank into the gel electrophoresis system.
Agarose gelTAE buffer
Well
TAE buffer
f. Observe bands appearing from (-) to (+) electrode
(-)
(+)
Moving Direction Weight
Heavier (move slower)
Lighter (move faster)
Just cover the surface of the gel
Bands of different dyes
S1 S2 S3
Avoidcontamination:• Changeanewtipevery time.Avoidgettingairintothebuffer:• Press to 1st stop ONLY and hold the plunger while
removing the pipette tip out of the buffer.
Lab2 FlowChart :IdentifyingarecombinantplasmidpARA-RMM PCRmastermix
A-rfp PlasmidAwithrfp gene(pARA-R)
A PlasmidA(pARA)
LD Loadingdye
M DNAladder
H2O Water
1xTAE 1X TAEbuffer
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• LabelthreePCRtubes 1,2and 3withyourgroupnumber.
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• Addreagentsaccording totheorder inTable2.1.• Gentlypipette upanddownseveraltimestomix.
Table2.1:Addition ofreagentstothePCRtubes
1 2 3
(a) PCRmastermix(MM) 23.0µL 23.0µL 23.0µL
(b) Plasmid A with rfp gene (A-rfp) 2.0µL
(c) Plasmid A (A) 2.0µL
(d) Water(H2O) 2.0µL
Total volume 25.0µL 25.0µL 25.0µL
Avoidcontamination:• Changeanewtipeverytimeafteraddingasolution.
Lab2.1:CheckingplasmidwithPCR
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• Return allPCRtubestotheiceimmediately.
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• Gentlytapthebottoms ofthePCR tubesorcentrifuge thetubesifthere arebubbles.
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• TakeyouricecupwithPCRtubestoyourteacher.• TransferyourPCRtubesfromtheiceintothe
thermocycler.
minipcr
1 2 3
Inkcancomeoff thetopofthetubeinthethermocycler(PCRmachine).
Avoidwarmingreagents:• Tubesmustbekeptcoldinice.• Holdthetubebytheupperrim.
Temperature(°C)
Time(sec)
Initialhold 4 IndefiniteInitialdenaturation 95 270
30cycles [
DenaturationAnnealingExtension
955368
303060
Finalextension 68 300
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• Thethermocycler hasbeenpre-programmed forthereaction.
Table2.2:PCRthermocycler programforABE
• Add2µLofloadingdye(LD)toeachofthethreePCRtubesandtothetubeswith DNAladder(M).
• Gentlypipetteupanddown severaltimestomix.
• Draw the location of the wells in your notebook.
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• Add 10µLof theDNAladder (M)and eachsamples(1,2,3) intodesignatedwells.
Avoidcontamination:• Changeanewtipevery time.Avoidgettingairintothebuffer:• Press to 1st stop ONLY and hold the plunger while
removing the pipette tip out of the buffer.
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• Aftertwoorthreeminutes, see if the bands aremovingtowards the positive electrode.
Makesurethewellsarenearthenegativeelectrode.10
Lab2.2:Confirmationbygelelectrophoresis
2.0 µL LD
1 2 3 M
Avoidcontamination:• Changeanewtipeverytimeafteraddingasolution.
M 1 2 3
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M 21 3
Agarose gelTAE buffer
Well
TAE buffer
• Pour 1x TAE buffer (1xTAE) into gel tank.• Put the gel tank into the gel electrophoresis
system.
~20 min
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TAE bufferAgarose gel
Just cover the surface of the gel10.0 µL
• Placethephotohoodonthegelelectrophoresis system• Turnonpowersupplyandpresstheon/offbutton.• Check ifbubbles form inthebufferatthe (-)electrode.
• After 20 minutes, observe the bands.• Record the location of bands in your notebook
(-)
(+)
Moving Direction Fragment
Size
Larger (move slower)
Smaller (move faster)
Bands shows different size of DNA fragment
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Lab3.1FlowChart:Cuttingthetwoplasmid(RestrictionDigestion)2.5x RB 2.5xRestriction Buffer
K PlasmidK(pKAN-R)
A PlasmidA(pARA)
RE Restriction Enzymes(BamHI& HindIII)
H2O Water
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• Label4newtubesasK+,K–,A+,andA–with classandgroup no..
2 • Addreagentsaccording totheorder inTable 3.1.
Table3.1:Addition ofreagentstotheK+,K–,A+,andA– tubes
Avoidcontamination:• Changeanewtipeverytimeafteraddingasolution.Mixwell:• Gentlypipetteupanddownthreetimes.
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• Spinthefourmicrofugetubes(K+,A+,K–,and A–)forfewseconds.
Balance theweight:• Distributethetubesevenly
Topoolthereagentsatthebottomofeachtube 4
• Incubate4tubesin37°Cwaterbathfor60mins.
Avoidnon-specific cutting:• Incubatenotlongerthan2hours
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• Aftertheincubation, store4tubesinthe–20°Cfreezer foruseinLab3.2.
‘+’meanspresent;‘–’meansabsent
K+ K– A+ A–
(a) Restriction buffer(2.5xRB) 4.0µL 4.0µL 4.0µL 4.0µL
(b) Plasmid K (K) 4.0µL 4.0µL
(c) Plasmid A (A) 4.0µL 4.0µL
(d) Restriction Enzymes(RE)andmix 2.0µL 2.0µL
(e) Water(H2O)andmix 2.0µL 2.0µL
TotalVolume 10 µL 10µL 10µL 10 µL
Lab3.2 FlowChart:Puttingtherfpgeneintotheplasmid(Ligation)K+ DigestedplasmidK(pKAN-R)
A+ DigestedplasmidA(pARA)
5xLigB 5xligationbufferLIG DNA ligase
H2O Water
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• PlacetheK+ andA+in70°Cwaterbathfor30mins.
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Table3.2:Addition ofreagentsLIGtubes
• Addreagentsdirectly intoLIG according totheorder inTable3.2.4
• LabelLIGwith classandgroupno..
• After30minutes, removeK+ andA+fromthewaterbath.
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LIG(with2.0µL ofDNALigase)
(a) DigestedplasmidA(A+) 4.0µL
(b) Digested plasmid K (K+) 4.0µL
(c) 5x ligation buffer (5x LigB) 3.0µL
(d) Water(H2O)andmix 2.0µL
TotalVolume 15 µL
Balance theweight:• Distributethetubesevenly
Poolthereagentsatthebottomofeachtube5
• SpintheLIG tubeforafewseconds. • IncubateLIG tube atroomtemperature overnight.
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• StoreK+ andA+ tubesinthe–20°CfreezerforuseinLab3.3.
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Avoidcontamination:• Changeanewtipeverytimeafteraddingasolution.Mixwell:• Gentlypipetteupanddownthreetimes.
LIG
Lab3.3FlowChart:ConfirmationbyGelElectrophoresisK- Nondigested plasmidK(pKAN-R)
K+ Digested plasmidK(pKAN-R)
A- Nondigested plasmidA(pARA)
A+ DigestedplasmidA(pARA)
LIG Ligatedplasmid
LD Loadingdye
H2O Water
M DNA ladder
1xTAE 1x TAEbuffer
1
• Labelfivenewmicrofuge tubes gelA–,gelA+,gelK–,gelK+ andgelLIGwith classandgroupno..
2 • Addreagentsaccording totheorder inTable3.3.• Pipette upanddownseveraltimestomix.
Table3.3:AdditionofreagentstothegelK–,gelK+,gelA–,gelA+,gelLIGand M tubes
gelK– gelK+ gelA– gelA+ gelLIG M (contain 8µLDNAladder)
(a) Water(H2O) 4.0µL 4.0µL 4.0µL 4.0µL 3.0µL
(b) Loading Dye (LD) 2.0µL 2.0µL 2.0µL 2.0µL 2.0µL 2.0µL
(c) Nondigested plasmid K (K–) and mix 4.0µL
(d) DigestedplasmidK(K+)andmix 4.0µL
(e) Nondigested plasmidA(A–)andmix 4.0µL
(f) DigestedplasmidA(A+)and mix 4.0µL
(g) Ligatedplasmid(LIG)andmix 5.0µL
Total volume 10 µL 10µL 10µL 10 µL 10 µL 10µL
Avoidcontamination:• Changeanewtipeverytimeafteraddingasolution.
‘gel’indicatesgelelectrophoresissamples
gel K–
gel K+
gel A–
gel A+
gel LIG
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• Return theLIGtube toyourteacher tostore inthe –20°C freezerforuse inLab4.
4 Poolthereagentsatthebottomofeachtube.
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• Pour1xTAE buffer (1x TAE) into gel tank.• Put the gel tank into the gel electrophoresis system.
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• Add 10µLoftheMandfivesamples(gelA–,gel A+,gelK–, gelK+ andgelLIG)into the wells
M gel K+ gel A+gel K– gel A– gel LIG
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Agarose gelTAE buffer
Well
TAE buffer
TAE bufferAgarose gel
~20 min
• Spinthesixtubes (gelA–,gelA+,gelK–, gelK+, gelLIGandM)forafewseconds.
Just cover the surface of the gel
Avoidcontamination:• Changeanewtipevery time.Avoidgettingairintothebuffer:• Press to 1st stop ONLY and hold the plunger while
removing the pipette tip out of the buffer.
• Aftertwoorthreeminutes, see if the bands aremovingtowards thepositive electrode.
• After 20 minutes, observe the bands.• Record the location of bands in your notebook
(-)
(+)
Moving Direction Fragment
Size
Larger (move slower)
Smaller (move faster)
Bands shows different size of DNA fragment
10.0 µLMakesurethewellsarenearthenegativeelectrode.
• Placethephotohoodonthegelelectrophoresis system• Turnonpowersupplyandpresstheon/offbutton.• Check ifbubbles form inthebufferatthe (-)electrode.
Which pipette to Use?
Use ASEPTIC techniques throughout the whole lab. 1
Label two tubes of E. coli cells with “–” and “+” and your group no. .
• Add 10 µL of LIG (or A-rfp) to “E. coli+”.• Gently flick the tube three times to mix.• Return the tube immediately to ice.
Keep E. coli– and E. coli+ on ice for 15 mins.
• Prepare three agar Petri plates—LB, LB/amp, and LB/amp/ara.
• Label the bottom of each platewith class & group no. .
Label LB/amp/ara plate as “E. coli+”. • Draw a line in the middle of LB andLB/amp plate.
• Label half of each plate “E. coli–” and theother half “E. coli+”.
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5a 5b 5c
Avoid warming cells: • Keep two tubes on ice.• Do not hold the bottom of the tubes.
Avoid contamination: • keep the plates closed while labelling
Write small on edge of the plate.
Part 2: Transformation (Heat Shock and Recovery)
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After 15-min incubation on ice, incubate the E. coli tubes in 42°C water bath for exactly 45 sec.
Immediately place the tubes back on ice for 2 mins.
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Incubate the E. coli tubes at room temperature (or 37°C) for 15 mins..
Avoid warming cells: • Carry tubes in the cup of ice to water bath.
Avoid contamination:• change a new tip every time after adding a solution.
Which pipette to Use?
Lab 4 Flowchart: Transforming Bacteria with Recombinant PlasmidLIG/A-rfp Ligated plasmid/ Plasmid A with rfp geneLB Luria BrothE. coli 50 µL of chilled competent E. coli cells x 2LB Plate contains Luria Broth (LB)LB/amp (one stripe) Plate contains Luria Broth (LB) and ampicillin (amp)LB/amp/ara (two stripes) Plate contains Luria Broth (LB), ampicillin (amp) and sugar arabinose (ara)
Part 1: Sample Preparation
• Add 150 µL of LB to the E. coli– and E. coli+ tubes• Gently flick it three times to mix.
10.0 µL LIG/A-rfp
E. coli +
LB LB/amp LB/amp/ara LB/amp LB LB/amp/ara
E. coli– E. coli+ E. coli+
45 sec.42º C
E. coli + E. coli –
E. coli– E. coli+
2 min.E. coli + E. coli –
150 µL LB
E. coli + E. coli –
37º C
15 min.E. coli + E. coli –
15 min.E. coli + E. coli –
Part 3: Spread the Cells on Plates for Incubation
10a, b & c 11a, b & c
• Suspend E. coli– cells by gently pump the pipette two orthree times.
• Add 50 µL of E. coli– cells to “E. coli–” on LB plate andLB/amp plate.
Use the same spreader to spread the E. coli– cells evenlyacross the entire “E. coli–” section on LB and LB/amp plate.
• Repeat step 10 for E.coli+.• Add 50 µL E. coli+ cells “E. coli+” on LB and LB/amp plates.• Add 100 µL E. coli+ cells to the LB/amp/ara plate.
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• Repeat step 11 for E.coli+.• Spread the E. coli+ cells evenly across the entire “E. coli+”
section on LB, LB/amp, and LB/amp/ara plates.
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Leave all plates right side up for 5 mins.
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• Tape all three plates together• Label the tape with class & group no.
Incubate the plates at 37°C upside down for 24–36 hours.
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Examine the plates and record the amount of growth on each half.
Which pipette to Use?
Avoid contamination:• Change a new tip every time after adding a solution.• Open lid just big enough to add the cells (like a clamshell)Avoid the cells slipping to another half of the plates:• Add the cells slowly to the section
Avoid contamination:• Hold the spreader by the handle. • Do not allow the bent end to touch any surface.• Open lid just big enough to add the cells (like a clamshell)
Prevent condensation from dripping on gels: • Incubate the plate upside down
50 µL E. coli –
LB E. coli – LB/amp
E. coli– E. coli+ E. coli– E. coli+
50 µL E. coli +
LB E. coli + LB/amp
E. coli– E. coli+ E. coli– E. coli+ E. coli+
5 min.
HandleSpreadingsurface
HandleSpreadingsurface
LB/amp/ara
100 µL E. coli +
37º C
24–36 hours
