Team:CityU HK/Protocol

Protocol

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.