Team:CityU HK/Protocol

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• PCR purification
• Plasmid extraction
• Gel purification
• Protein purification
• Quantitative RT-PCR
• Reverse transcription PCR
• RNA extraction
• ONPG assay
• Colony PCR

PCR Amplification Protocol

Using TIANGEN 2× Pfu PCR MasterMix

 

PCR Reaction Mixture

	Volume (μl) for 1 reaction	Master mix (μl) for 4.2 reactions
ddH2O	9.5	39.9
Pfu PCR MasterMix (2X)	12.5	52.5
F-primer (10 mM)	1	4.2
R-primer (10 mM)	1	4.2
Total	24	/

§   Add 1 μl of DNA template to 24 μl of master mix in each PCR tube.

Thermocycling Conditions

Step		Temperature (oC)	Time
Initial denaturation		94	3 minutes
Denaturation		94	30 seconds
Annealing		55	30 seconds
Extension (This process is repeated for 32 cycles)		72	2 minutes*
Final extension		72	5 minutes
Hold		12	∞

*Depending on the size of amplicon (1,000 bp/minute)

 

PCR Amplification Protocol

Using TIANGEN 2× Taq Plus PCR MasterMix

 

PCR Reaction Mixture

	Volume (μl) for 1 reaction	Master mix (μl) for 25 reactions
ddH2O	7.4	185
Taq Plus PCR MasterMix (2X)	10	250
F-primer (10 mM)	0.8	20
R-primer (10 mM)	0.8	20
Total	19	/

§   Add 1 μl of each DNA template to 19 μl of master mix in each PCR tube.

Thermocycling Conditions

Step		Temperature (oC)	Time
Initial denaturation		94	3 minutes
Denaturation		94	30 seconds
Annealing		55	30 seconds
Extension (This process is repeated for 32 cycles)		72	1 minute*
Final extension		72	5 minutes
Hold		12	∞

*Depending on the size of amplicon (1,000 bp/minute)

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Purification of PCR Products Protocol

Using TIANGEN^® TIANquick Midi Purification Kit (DP204)

 

[1]        Add 500 μl of Buffer BL to a CB2 spin column in a collection tube. Centrifuge for 1 minute at 12,000 rpm (~13,400 x g). Discard the flow-through.

[2]        Add 5 volumes of Buffer PB to 1 volume of nucleic acid solution in a 1.5 ml microcentrifuge tube. Mix gently.

[3]        Transfer the mixture to the spin column and incubate at room temperature for 2 minutes. Centrifuge for 1 minute at 12,000 rpm (_~13,400 x g). Discard the flow-through.

[4]        Add 600 μl of Buffer PW to the spin column and centrifuge for 1 minute at 12,000 rpm (_~13,400 x g). Discard the flow-through. Repeat.

[5]        Centrifuge the empty spin column for 2 min.

[6]        Place the spin column in a clean 1.5 ml microcentrifuge tube. Add 30 μl of Buffer EB or ddH₂O (prewarmed at 60^oC) to the center of the membrane. Incubate at room temperature for 2 minutes. Centrifuge at 12,000 rpm (_~13,400 x g) for 2 minutes.

[7]        Repeat step 6 by using the flow-through (DNA) in the 1.5 ml microcentrifuge tube.

[8]        Store the DNA at 4^oC or –20^oC.

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Plasmid Extraction Protocol

Using TIANGEN^® TIANprep Mini Plasmid Kit (DP103)

 

[1]        Add 500 μl of Buffer BL to spin column CP3 with collection tube to activate the DNA-binding membrane. Centrifuge at 12,000 rpm (_~13,400 x g) for 1 minute. Discard the flow-through.

[2]        Harvest a total of 3 ml of bacterial cells in a 1.5 ml microcentrifuge tube by centrifuging 1.5 ml bacterial cells at 12,000 rpm (_~13,400 x g) for 1 minute and repeat.

[3]        Remove the supernatant. Resuspend the cell pellet in 250 μl Buffer P1 until no cell crumbs can be seen.

[4]        Add 250 μl of Buffer P2 and mix thoroughly by inverting the tube 6 to 8 times. (No vortexing)

[5]        Add 350 μl of Buffer P3 and immediately invert the tube 6 to 8 times. (No vortexing)

[6]        Centrifuge the tube at 12,000 rpm (_~13,400 x g) for 10 minutes.

[7]        Apply the supernatant to the activated spin column and centrifuge for 1 minute at 12,000 rpm (_~13,400 x g). Discard the flow-through.

[8]        Add 500 μl of Buffer PD to the spin column and centrifuge for 1 minute at 12,000 rpm (_~13,400 x g). Discard the flow-through.

[9]        Add 600 μl of Buffer PW to the spin column and centrifuge for 1 minute at 12,000 rpm (_~13,400 x g). Discard flow-through. Repeat.

[10]    Spin the empty spin column for 2 minutes.

[11]    Place the spin column in a clean 1.5 ml microcentrifuge tube. Add 50 μl of Buffer EB or ddH₂O (prewarmed at 60^oC) to the center of the membrane. Incubate at room temperature for 2 minutes. Centrifuge for 2 minutes at 12,000 rpm (_~13,400 x g).

[12]    Repeat step 11 by using the flow-through (DNA) in the 1.5 ml microcentrifuge tube.

[13]    Store the DNA at 4^oC or –20^oC.

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Purification of DNA from Gel Protocol

Using TIANGEN^® TIANgel Maxi DNA Purification Kit (DP210)

 

[1]        Excise the desired DNA fragment from the agarose gel with a clean and sharp scalpel. Slice the gel piece into small pieces.

[2]        Transfer the pieces into a microcentrifuge tube.

[3]        Add 3 volumes of Buffer PN to 1 volume of gel pieces in a 1.5 ml microcentrifuge tube and mix well. Incubate the tubes in a 50^oC waterbath until all gel pieces have completely dissolved.

[4]        During the incubation, add 500 μl of Buffer BL to a CA3 spin column. Centrifuge at 12,000 rpm (~13,400 x g) for 1 minute and discard the flow-through.

[5]        Apply the DNA mixture to the spin column (£ 800 μl) and incubate at room temperature for 2 minutes. Centrifuge 1 minute at 12,000 rpm (~13,400 x g). Discard the flow-through. Repeat to transfer the remaining DNA mixture if necessary.

[6]        Add 600 μl of Buffer PW to the spin column and centrifuge for 1 minute at 12,000 rpm (~13,400 x g). Discard the flow-through. Repeat.

[7]        Spin the empty spin column for 2 minutes.

[8]        Place the spin column in a clean 1.5 ml microcentrifuge tube. Add 30 μl of Buffer EB or ddH₂O (warmed at 60^oC) to the center of membrane. Incubate at room temperature for 2 minutes. Centrifuge for 2 minutes at 12,000 rpm (~13,400 x g).

[9]        Repeat step 8 by using the flow-through (DNA) in the 1.5 ml microcentrifuge tube.

[10]    Store the DNA at 4^oC or –20^oC.

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Colony PCR (analysis) Protocol

Using TaKaRa Ex Taq^® DNA Polymerase

 

Part I: Cell lysate (template) preparation

[1]        Select well isolated colonies on LB plates for PCR amplification. Prepare a PCR tube for each colony.

[2]        Pick colony with a sterile pipette tip and resuspend colony in 5μl of sterile ddH₂O.

[3]        Heat PCR tubes in the thermocycler at 98^oC for 10 minutes to lyse the cells. And centrifuge the lysate at 14,000 rpm for 1 minute.

Part II: PCR

PCR Reaction Mixture

	Volume of reaction mix (μl) for 1 reaction	Volume of reaction mix (μl) for 12 reactions
Ex Taq polymerase (5 units/μl)	0.075	0.9
Taq PCR buffer (10X)	1.5	18
dNTPs (10 mM)	1.2	14.4
VF2 primer (10 mM)	0.3	3.6
VR primer (10 mM)	0.3	3.6
ddH2O	10.625	127.5
Total	14	168

§   Mix 1 μl of DNA template (from step 3) with 14 μl of master mix in a PCR tube.

Thermocycling Conditions

Step		Temperature (oC)	Time
Initial denaturation		98	2 minutes
Denaturation		98	30 seconds
Annealing		55	30 seconds
Extension (This process is repeated for 32 cycles)		72	2 minutes*
Final extension		72	1 minute
Hold		4	∞

*Depending on the size of amplicon (e.g. 2 minutes for 1 to 2 kb)

 

Colony PCR (analysis) Protocol

Using NEB Quick-Load^® Taq 2X Master Mix

 

Part I: Cell lysate (template) preparation

[1]        Select well isolated colonies on LB plates for PCR amplification. Prepare a PCR tube for each colony.

[2]        Pick colony with a sterile pipette tip and resuspend colony in 5μl of sterile ddH₂O.

[3]        Heat PCR tubes in the thermocycler at 98^oC for 10 minutes to lyse the cells. And centrifuge the lysate at 14,000 rpm for 1 minute.

Part II: PCR

PCR Reaction Mixture

	Volume of reaction mix (μl) for 1 reaction	Volume of reaction mix (μl) for 10 reactions
Quick-Load Taq Master Mix (2X)	7.5	75
VF2 primer (10 mM)	0.075	0.75
VR primer (10 mM)	0.075	0.75
ddH2O	6.35	63.5
Total	14	140

§   Mix 1 μl of DNA template (from step 3) with 14 μl of master mix in a PCR tube.

Thermocycling Conditions

Step		Temperature (oC)	Time
Initial denaturation		95	30 seconds
Denaturation		95	30 s
Annealing		55	30 seconds
Extension (This process is repeated for 32 cycles)		68	5 minutes*
Final extension		68	5 minutes
Hold		4	∞

*Depending on the size of amplicon (e.g. 5 minutes for 4 to 5 kb)

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Genomic DNA Extraction Protocol

QIAGEN^® DNeasy 96 Blood & Tissue Kit

 

[1]        Harvest cells by centrifugation at 5000 x g (7500 rpm) for 10 minutes. Discard supernatant.

[2]        Resuspend cell pellet in 180 µl Buffer ATL.

[3]        Add 20 µl proteinase K to the mixture. Mix thoroughly by vortex, and incubate in a 56°C water bath until the tissue has completely lysed. Vortex occasionally during incubation.

[4]        Add 4 µl RNase A (100 mg/ml), mix by vortexing and incubate at room temperature for 30 minutes.

[5]        Vortex for 15 seconds.

[6]        Add 200 µl of Buffer AL to the sample, and mix thoroughly using a vortex.

[7]        Add 200 µl of ethanol (96–100%), and mix again thoroughly by vortex.

[8]        Pipette the mixture from step 7 (including any precipitate) into the DNeasy Mini spin column placed in a 2 ml collection tube. Centrifuge at 6000 x g (8000 rpm) for 1 minute. Discard the flow-through and collection tube.

[9]        Place the DNeasy Mini spin column in a new 2 ml collection tube, add 500 µl of Buffer AW1, and centrifuge for 1 minute at 6000 x g (8000 rpm). Discard the flow-through and collection tube.

[10]    Repeat step 9 with centrifuge time 3 minutes at 20,000 x g (14,000 rpm) to dry the DNeasy membrane. Discard the flow-through and collection tube.

[11]    Place the DNeasy Mini spin column in a clean 1.5 ml microcentrifuge tube, and pipette 200 µl of Buffer AE directly onto the DNeasy membrane. Incubate at room temperature for 1 minute and then centrifuge for 1 min at 6000 x g (8000 rpm) to elute the DNA.

[12]    Repeat the elution in step 11 to recover more DNA.

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Protein Extraction Protocol

BioVision^® EZLys^TM Bacterial Protein Extraction Reagent

 

[1]        Collect cells by centrifugation at 16,000g for 10 minutes in a pre-weighed centrifuge tube. Remove as much liquid as possible. Determine the net weight of the cell pellet.

[2]        Resuspend the pellet in at least 4 ml of EZLys reagent (pre-warmed to room temperature) per 1g of wet cell paste by pipetting and/or gentle vortex.

[3]        Incubate the cell suspension by shaking or slowly mixing for approximately 5 minutes.

[4]        Remove the insoluble cell debris by centrifugation at 16,000g for 20 minutes at 4^oC. The supernatant containing the soluble proteins can now by analyzed or further purified by using a Ni-NTA column or stored at -20^oC for future use.

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ONPG Assay Protocol

 

[1]        Incubate the E. coli culture in 10 ml of LB broth overnight at 37^oC with shaking.

[2]        Measure the OD₆₀₀ of the overnight culture following a 10-fold dilution.
(Dilute culture with LB broth and use LB broth as blank)

[3]        Transfer 1 ml of E. coli to a new glass test tube.

[4]        Add 1 ml of Z buffer to the tube.

[5]        Add 40 μl of chloroform and 20 μl of 0.1% SDS to the tube. Mix thoroughly.

[6]        Incubate the tube in a 37^oC water bath for 10 minutes. Mix vigorously.

[7]        Add 0.4 ml of ONPG (4 mg/ml) to the tube and shake for a few times.

[8]        Incubate the tube in a 37^oC water bath for 15 minutes.

[9]        Add 1 ml of 1 M Na₂CO₃ to the tube to stop the reaction.

[10]    Transfer 1 ml of supernatant from the tube to a cuvette.

[11]    Measure the OD₄₂₀ of the supernatant.
(Dilute with LB broth if necessary and use 0.4 ml ONPG + 2 ml Z buffer + 1 ml Na₂CO₃ as blank)

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RNA Extraction Protocol

Using TaKaRa^® MiniBEST Universal RNA Extraction Kit

 

[1]        Harvest 500 μl of bacterial cells in a 1.5 ml microcentrifuge tube by centrifugation at 12,000 rpm (_~13,400 x g) for 1 minute at 4^oC.

[2]        Remove the supernatant. Resuspend the cell pellet in 200 μl of freshly prepared lysozyme (0.5 mg/mL in Tris/EDTA Buffer) solution and ensure no cell clumps can be seen. Incubate at room temperature for 15 minutes.

[3]        Add 600 μl of Buffer RL and 12 μl of 50X DTT Solution. Mix thoroughly. Incubate the cell lysate at room temperature for 2 minutes.

[4]        Apply the cell lysate to a gDNA Eraser spin column with a 2 ml collection tube. Centrifuge at 12,000 rpm (_~13,400 x g) for 1 minute.

[5]        Discard the gDNA Eraser Spin Column. Add an equal volume of 70% ethanol (812 μl) to the flow-through in the 2 ml collection tube. Mix thoroughly.

[6]        Transfer the mixture to an RNA spin column with 2 ml collection tube. (If the volume of the mixture is more than 600 μl, apply the mixture by dividing into several aliquots). Centrifuge at 12,000 rpm (_~13,400 x g) for 1 minute. Discard the flow-through.

[7]        Add 500 μl Buffer RWA to the RNA spin column. Centrifuge at 12,000 (_~13,400 x g) for 30 seconds. Discard the flow-through.

[8]        Add 600 μl Buffer RWB to the RNA spin column. Centrifuge at 12,000 (_~13,400 x g) for 30 seconds. Discard the flow-through.

[9]        Prepare the DNase I mixture in a 1.5 ml microcentrifuge tube by adding 5 μl 10X DNase I Buffer, 4 μl Recombinant DNase and 41 μl RNase free H₂O. Mix gently by inverting the tube.

[10]    Add 50 μl of the DNase I mixture to the center of membrane. Incubate at room temperature for 15 minutes.

[11]    Add 350 μl RWB to the RNA spin column. Centrifuge at 12,000 rpm (_~13,400 x g) for 30 seconds. Discard the flow-through.

[12]    Repeat step 8.

[13]    Centrifuge the empty spin column at 12,000 rpm (_~13,400 x g) for 2 minutes.

[14]    Place the RNA spin column in a new 1.5 ml RNase free collection tube. Add 30 μl of RNase-free water to the center of membrane. Incubate at room temperature for 5 minutes. Centrifuge for 2 minutes at 12,000 rpm (_~13,400 x g).

[15]    Repeat step 14 by using the flow-through (RNA) in the 1.5 ml RNase free collection tube.

[16]    Store the RNA at -80^oC.

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Reverse-transcription PCR Protocol

Using TaKaRa PrimeScript™ RT reagent Kit with gDNA Eraser

 

Part I: Removal of genomic DNA in RNA samples

Digestion of genomic DNA

	Volume (μl) for 1 reaction	Master mix (μl) for 8 reactions
gDNA Eraser Buffer (5X)	2.0	16
gDNA Eraser	1.0	8.0
Total	3.0	24

§   Add 1 µg of each total RNA template to 3 μl of master mix in each PCR tube

§   Add an appropriate volume of RNase-free sterile dH₂O to yield a final volume of 10 μl in each PCR tube

§   Place the tubes inside the thermocycler under 37^oC for 2 minutes, followed by 85^oC for 5 seconds.

Part II: Reverse-transcription reaction

Reverse-transcription reaction

	Volume (μl) for 1 reaction	Master mix (μl) for 8 reactions
PrimeScript Buffer 2 (5X)	4.0	32
PrimeScript RT Enzyme Mix I	1.0	8.0
RT Primer Mix	1.0	8.0
RNase Free dH2O	4.0	32
Total	10.0	80.0

§   Add 10.0 μl of reaction solution from step 1 to 10.0 μl of master mix in each PCR tube

§   Place the tubes inside the thermocycler under 37^oC for 2 minutes, followed by 85^oC for 5 seconds.

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Real time PCR Protocol

Using Promega GoTaq® qPCR Master Mix

 

Part I: Standard Curve Construction

[1]        Prepare 4 concentrations of cDNA template by serially diluting a standard RT reaction product as follows:

Dilution	Recipe
A (1/5)	20 μl RT reaction product + 80 μl water
B (1/50)	10 μl Dilution A + 90 μl water
C (1/500)	10 μl Dilution B + 90 μl water
D (1/5000)	10 μl Dilution C + 90 μl water

 

[2]        Add 2 μl of each concentration (in triplicate) to appropriate wells on a 96-well PCR plate:

	Triplicate
Conc.	1	2	3
A	○	○	○
B	○	○	○
C	○	○	○
D	○	○	○

 

[3]        Prepare a PCR master mix as follows:

	Volume (μl) for 1 reaction	Master mix (μl) for 13 reactions
GoTaq® qPCR Master Mix (2X)	5.0	80
SYBP Green Supermix	0.1	1.3
10 μM Forward primer	0.18	2.34
10 μM Reverse primer	0.18	2.34
Water	2.54	33.02
Diluted cDNA	2.0	(pre-add to well)
Total	10	130

[4]        Mix a 8 μl PCR master mix with each cDNA aliquot by gently pipetting up and down several times

[5]        Seal the 96-well plate with an optical tape. Spin down all droplets by centrifugation at 3,000 rpm for 2 minutes.

Part II: Gene expression measurements

[1]        Dilute RT product 1 in 5 times as follows:
à20 μl RT reaction product + 80 μl water
If the expression of the target gene is low, prepare a less diluted sample of the RT-product for analysis.

[2]        Add 2 μl of the diluted RT product (in triplicate) to appropriate wells of a 96-well PCR plate.

[3]        Prepare a PCR master mix as follows:

	Volume (μl) for 1 reaction	Master mix (μl) for 4 reactions
GoTaq® qPCR Master Mix (2X)	5.0	20
SYBP Green Supermix	0.1	0.4
10 μM Forward primer	0.18	0.72
10 μM Reverse primer	0.18	0.72
Water	2.54	10.16
Diluted cDNA	2.0	(pre-add to well)
Total	10	40

 

[4]        Mix 8 μl PCR master mix with each cDNA aliquot by gently pipetting up and down several times

[5]        Seal the 96-well plate with an optical tape. Spin down all droplets by centrifugation at 3,000 rpm for 2 minutes.