Team:Hong Kong-CUHK/Laboratory/protocol

Field: Laboratory

Block: Protocols and experiments

 

Protocols Contents

1. Cloning

1.1 PCR Amplification of the gene

1.2 Double Digestion

1.3 PCR Purification

1.4Agarose Gel electrophoresis

1.5 Ligation

1.6 Gel Extraction

1.7 Bacterial Transformation

1.8 Inoculation

1.9 Extraction of Plasmid DNA

2. Electrodesfunctional test

3. Bacteria light-couple electricity generation

4. Measuring voltage vs. sodium chloride concentrationrelation of mixing entropy electrode

5. Cell growth optimization

5.1 Preparation of growth medium

5.2 Inoculation

5.3 Salt concentration test

6. Measurement ofchloride concentration in bacterial lysate using fluorescent dye N-(ethoxycarbonylmethyl)-6-methoxyquinoliniumbromide (MQAE)

7. Excitation ofbacteria to trigger the halorhopsin

8. Controlledbacterial cell movement measurement

9. Laser excitation protocol

9.1 General, wavelength

9.2 Laser power

9.3 excitation length

 

 

Cloning

PCR Amplification of the gene

 

- Reagents

1) PCR SuperMix High Fidelity

22 U/ml DNA polymerase mixture in:

- 66 mM Tris-SO₄ (pH 9.1 at 25°C)

- 19.8 mM (NH₄)₂SO₄

- 2.2 mM MgSO₄

- 220 μM dGTP

- 220 μM dATP

- 220 μM dTTP

- 220 μM dCTP

- stabilizers

2) Forward and Reverse Primer

3) DNA template

 

- Protocols

PCR Assembly from Reaction Components:

1) Set up reaction tubes on ice.

2) Add the following components in any order to each reaction tube:

a) 45 μl PCR SuperMix High Fidelity

b) Primer solution (200 nM finalconcentration of each is recommended)

c) Template DNA solution (Total volume ofprimer and template solutions added can be 0.5-5 μl)

3. Mix contents of tubes and cover with mineral or silicone oil.Depending on the model of thermal cycler used, mineral or silicone oil may notbe necessary.

4) Cap tubes and load thermal cycler at 94°C.

5) Start cycling program file

 

*Thefollowing general procedures are suggested as guidelines for use of PCRSuperMix High Fidelity. Assembly of the reactions on ice from pre-chilledcomponents is recommended. Final reaction volumes described below areapproximately 50 μl. Reaction size may be altered to suit user preferences.

 

**RecommendedCycling Program

1cycle of:  94°C for 30 seconds(Pre-amplification denaturation)

30–35cycles of:

 94°C for 30 seconds (Denaturation)

 55–68°C for 30 seconds (Annealing)

 68–72°C for 60 seconds per kb target length(Extension)

 Use of 68°C during extension step of PCR isstrongly recommended for fragments > 5 kb in length

 

Double Digestion

 

- Reaction mixtures

Total volume 50μl

37.6μl Insert/Vector +ddH₂O_*

5μl 10X Buffer3

5μl 10X BSA

1.2μl Enzyme 1

1.2μl Enzyme 2

_*Water is addedfirst and the template the last

 At least 200ng DNA should be added

 

- Protocol

Reaction mixture at 37^oc for 2 hours

Buffer Chart

or http://www.neb.com/nebecomm/tech_reference/restriction_enzymes/buffer_activity_restriction_enzymes.asp

 

PCR Purification

 

- *Protocol

1) Add5 volumes of Buffer PB to 1 volume of the PCR sample and mix. It is not        necessary to remove mineral oil orkerosene.

2) If pHindicator I has beein added to Buffer PB, check that the color of the mixtureis yellow. If the color of themixture is orange or violet, add 10 µl of 3 M sodium acetate, pH 5.0, and mix.The color of the mixture will turn to yellow.

3)Place a QIAquick spin column in a provided 2 ml collection tube.

4) Tobind DNA, apply the sample to the QIAquick column and centrifuge for 30–60s.

5)Discard flow-through. Place the QIAquick column back into the same tube.

6) Towash, add 0.75 ml Buffer PE to the QIAquick column and centrifuge for 30–60 s.

7)Discard flow-through and place the QIAquick column back in the same tube. Centrifugethe column for an additional 1 min. Protocol

8)Place QIAquick column in a clean 1.5 ml microcentrifuge tube.

9) Toelute DNA, add 50 μl Buffer EB (10 mM Tris·Cl, pH 8.5) or water (pH 7.0–8.5) tothe center of the QIAquick membrane and centrifuge the column for 1 min.Alternatively, for increased DNA concentration, add 30 μl elution buffer to thecenter of the QIAquick membrane, let the column stand for 1 min, and thencentrifuge.

10) Ifthe purified DNA is to be analyzed on a gel, add 1 volume of Loading Dye to

5volumes of purified DNA. Mix the solution by pipetting up and down before loadingthe gel.

IMPORTANT: Ensure that the elution buffer is dispensed directly onto theQIAquick

membrane for complete elution of bound DNA. The average eluate volume is48 µl

from 50 µlelution buffer volume, and 28 µl from 30 µl elution buffer.

*Protocol adopted fromQIAquick PCR purification kit protocol

 

 

 

Agarose Gel electrophoresis

 

- Set gel

1) Dissolve 0.55g Agarose into 55ml 0.5X TBEbuffer.

2) Microwave for 1 minute.

3) Cool it down using running water for 1 minute.

4) Add 1μl GelRed

5)Pour the solution to tightened tank with gates and gel comb and allow it tosolidify.

6)Transfer the gel to gel tank once it is set hard.

 

-Run gel

1)Orientthe gel with wells facing the black negative electrode. Check if the gel iscovered by TBE buffer in the tank. If not, add TBE buffer to cover it to about1mm.

2) Mix loadingdye and the insert/plasmid before adding to the wells. For example, if the DNAwe have got is 45μl, and the loading dye we have got is 10X, then add 5μl ofloading dye to the samples. Mixture should be in blue.

3)Torun gel, add all samples to the wells of gel. Then add 1kb DNA ladder to aseparate well. 1μl should be enough for detection under UV.

4)Connectthe electrodes to the power supply with correct colour. Apply power supply with120V. Check if there are bubbles on the negative electrodes.

5) Allow it torun for about 20 - 30 mins. To avoid running the band off the gel, check theyellow band, which is the smallest fragments, should stay on the gel.

 

*Gel Extraction

 

1. Excise the DNA fragment from the agarose gel with a clean, sharp scalpel.

2. Weigh the gel slice in a 2 ml centrifuge tube. Add 3 volumes ofBuffer QG to

1 volume of gel (100 mg ~ 100 μl).

3. Incubate at 50°C until the gel slice has completely dissolved (around10  min). Mix by vortexing the tube every2 mins during the incubation.

4. After the gel slice has dissolved completely, check that the color ofthe  mixture is yellow. If the color ofthe mixture is orange or violet, add 5-10 μl of 3 M sodium acetate, pH 5.0, andmix until it turn to yellow.

5. Add 1 gel volume of isopropanol to the sample and mix (for DNAfragments  <500 bp and >4 kb).

6. Place a QIAquick spin column in a provided 2 ml collection tube.

7. To bind DNA, apply the sample to the QIAquick column, and centrifugefor 30 s. (The maximum volume of the column reservoir is 800 μl. For samplevolumes of more than 800 μl, simply load and spin again).

8. Discard flow-through and place QIAquick column back in the same  collection tube. Collection tubes are re-usedto reduce plastic waste.

9. Add 0.5 ml of Buffer QG to QIAquick column and centrifuge for 30 s toremove all traces of agarose.

10. To wash, add 0.75 ml of Buffer PE to QIAquick column, stand for 2mins and centrifuge for 30 s.

11. Discard the flow-through and centrifuge the QIAquick column for anadditional 30 s and air-dry for 2 mins (This step can ensure all ethanol isremoved and the column is NOT over-dry)!

12. Place QIAquick column into a clean 1.5 ml microcentrifuge tube.

 

13. To elute DNA, add 35 μl of 50 oC ddH2O to the center of the QIAquick

membrane, wait2 mins and centrifuge the column for 2 mins at maximum speed.

*extracted from CUHK iGEM2010 protocol

 

Ligation

 

- Reaction mixture

Total volume 20μl

2μl 10X Ligationbuffer

1μl T4 DNA ligase

14μl Vector

3μl insert

 

- Protocol

Allow the reaction to be taken place at 16^oC overnight, or 22^oCfor 1 hour.

 

Bacterial Transformation

 

- Transformation ofE. coli (DH5α) with plasmid DNA

1) Thaw competent cell on ice.

2) Add 50 ~ 100 ng DNA to competent cell culture.

3) Put in ice for 30 min.

4) Heat shock at 42^oC for 1 minute.

5) Put in ice for 2 min.

6) Add 1 ml LB broth medium.

7) Incubate at 37^oC for 90 min with shaking (~ 250 rpm).

8) Spread plate (with suitableantibiotics):

9) Spin down the remaining cells and discard large amountsupernatant (1ml), thenresuspend cells.

10) Incubate at 37^oC overnightuntil colonies are obtained.

 

Inoculation

 

- Protocol

1) Pick colonies & culture in 2~ 3 ml LB broth with antibiotics.

2) Incubate at37^oC for 12-16 hours with shaking at 250rpm.

 

Extractionof Plasmid DNA (Miniprep)

 

- Protocol

1. Resuspend pelleted bacterial cells in 250 µl Buffer P1 and transferto a micro-

centrifuge tube.

2. Add 250 µl Buffer P2 and mix thoroughly by inverting the tube 4–6times and incubate 2 min at room temperature. Do not vortex!

3. Add 350 µl Buffer N3 and mix immediately and thoroughly by invertingthe tube

4–6 times.

4. Centrifuge for 10 min at 13,000 rpm in a table-top microcentrifuge. Acompact white pellet will form.

5. Apply the supernatants from step 4 to the QIAprep spin column

6. Centrifuge for 30–60 s. Discard the flow-through.

7. Recommended: Wash the QIAprep spin column by adding 0.5 ml Buffer PBand

centrifuging for 30–60 s. Discard the flow-through.

8. Wash QIAprep spin column by adding 0.75 ml Buffer PE and centrifugingfor

30–60 s.

9. Discard the flow-through, and centrifuge for an additional 1 min toremove residual

wash buffer.

10. Place the QIAprep column in a clean 1.5 ml microcentrifuge tube.

add 50 µl Buffer EB (10 mM Tris·Cl, pH 8.5) or water to the center ofeach QIAprep

spin column, let stand for 1 min, and centrifuge for 1 min to elute theDNA.

Electrodesfunctional test

 

- Protocol

1) Put electrodes to 100 ml doubledistilled water, measure the voltage by voltmeter between two electrodes untilthe reading achieve steady.

2) Put electrodes to 100 ml, 0.4 M sodiumchloride solution. Parallel connect a voltmeter and an ammeter to between twoelectrodes to measure voltage and current. Take the readings every 30 seconds.

3) When the voltage reading achieve steady,replace sodium chloride solution with double distilled water. While keep takingreadings every 30 seconds.

4) Putthe electrodes to fresh water. Keep taking the readings every 30 seconds untilthe voltage achieve steady.

5)Plot voltage vs. time and current vs. time. Calculate the power for every timepoint. Estimate the total electric work.

 

Bacterialight-couple electricity generation

 

- Protocol

1) Parallel connect the electrodes with avoltmeter and ammeter. Put two electrodes to 50 ml, O.D. 600 bacteria solutionand shut down all the light.

2) Wait the voltage achieve steady, startrecord the voltage and current every 30 seconds, for totally 5 minutes.

3) Use light source directly illuminate onthe bacteria solution, take the reading every 30 seconds for totally 5 minutes.

4) Remove the light source and take thereading every 30 seconds, for totally 5 minutes.

5) Wait until the voltage become steady,start record the voltage and current every 30 seconds for totally 15 minutes.This step is for control.

6) Plot voltage vs. time and current vs.time for measurement and control respectively. Calculate the power and estimatetotal electric work.

 

Measuringvoltage vs. sodium chloride concentration relation of mixing entropy electrode

 

- Protocol

1) Put electrodes to double distilled waterand measure the voltage between to electrodes until the reading become steady.

2) Put electrodes to 100 ml 0.05 M sodiumchloride concentration and wait the reading achieve steady. Record the steadyvoltage.

3) Add sodium chloride powder to make thefinal concentration according to the table below. Record the steady voltage foreach concentration.

Final concentration (M)	Total volume (ml)	Weight of total sodium chloride (g)	Weight of added sodium chloride (g)
0.10	100	0.58	0.29
0.15	100	0.88	0.29
0.20	100	1.17	0.29
0.25	100	1.46	0.29
0.30	100	1.75	0.29
0.35	100	2.05	0.29
0.40	100	2.34	0.29
0.45	100	2.63	0.29
0.50	100	2.92	0.29
0.55	100	3.21	0.29
0.60	100	3.51	0.29

 

4) Put the electrode in double distilledwater until the reading become steady to wash away sodium chloride. Repeat thisstep for 2 times. Then take the reading for concentration 0 M.

5) Repeat the whole process for two moretimes.

 

Cell growth optimization

Preparation of growth medium (500mlLB (Luria Broth), 500ml 1M NaCl LB and 500ml 1M KCl LB)

 

- Protocol

1) Weigh 12.5gLB powder and add the powder into a 500ml bottle.

2) Add 500ml distilled water to the bottle.

3) Autoclave the solution.

4) 29.22g NaCland 37.25g KCl is added respectively to prepare 500ml 1M NaCl LB and 500ml 1MKCl LB.

 

Inoculation

 

-Protocol

1) Add 5ml LB solution to a falcon.

2) Add 5ul antibiotic K.

3) Add 2.5ul IPTG.

4) Pick a single colony from plate spread with DH5α transformedwith pET27b HR and add it into the falcon.

5) Put the start culture inside the incubator at 37℃ and shaking 200rpm.

6) Collect thestart culture the other day.

 

Salt concentration test

 

- Protocol

 

A. Preparation of 6 sets of solution:

0.1-1M NaCl with IPTG

0.1M-1M NaCl without IPTG

0.1M-1M KCl with IPTG

0.1M-1M KCl without IPTG

0M salt with IPTG

0M salt without IPTG

1) Prepare the medium as shown:

[Salt]/M	0	0.1	0.2	0.3	0.4	0.5	0.6	0.7	0.8	0.9	1.0
LB +1M salt/ml	0	0.4	0.8	1.2	1.6	2.0	2.4	2.8	3.2	3.6	4
LB/ml	4.0	3.6	3.2	2.8	2.4	2.0	1.6	1.2	0.8	0.4	0

 

2) Add 2ul IPTG(only applicable to those with IPTG,otherwise go to step 3).

3) Add 40ul of DH5α transformedwith pET27b HR start culture into each tube.

4) Use a test tube rack to hold all the tubes andput the rack inside the incubator at 37℃. andshaking 200rpm(?).

 

 

B.Measurement of absorbance

1) Set the wavelength of spectrophotometer to 600nm.

2) Set blank with 1ml LB solution.

3) Pipette up and down to mix the solution by P1000pipette.

4) Transfer 1ml solution to a cuvette.

5) Record the OD600.

 

C. Measurement of bacterial growth curves

1) Inoculate the cells overnight.

2) Autoclave conical flasks with 100 ml LB solution.

3) Measure the OD600 and calculate the relativeamount of bacteria needed and add into the conical flasks (usually 1%inoculums).

4) Measure the OD600 after adding the bacteria intothe conical flasks. This is time 0.

Measure the OD600 every half hour for eight hours.

 

Measurementof chloride concentration in bacterial lysate using fluorescent dye N-(ethoxycarbonylmethyl)-6-methoxyquinoliniumbromide (MQAE)

 

- Protocol

1) Culture the cells overnight.

2) Centrifuge for 1 min in maximum speed andcollect the cell pellet.

3) Lyse the cells by mixing well with 2% SDSand boil at 99℃for 5 min.

4) Centrifuge for 1 min in maximum speed andobtain supernatant .

5) Dilute the supernatant to desiredconcentrations.

6) Add 95ul of each sample to 96well-microplate.

7) Add 5ul 0.5mM MQAE dissolved in DMSOafterwards.

8) Incubate for 1 hour at 37℃.

9)Measure the fluorescence by microplatereader using 350 nm absorption wavelength and 460 nm emission wavelength

Later found that the halorhodopsin’s colour affect the measurement ofthe MQAE fluorescence, need to change to centrifuge for 1 h in maximum speed at4℃and obtain supernatant after boiling with SDS to separate the halorhodopsinfrom the cell lysate.

 

Excitation of bacteria to trigger thehalorhopsin

 

-Protocol

1) Usealuminum foil to wrap start cultures.

2) Excite thebacteria using either 405, 530, 670 nm lasers with the confocal microscope withlive cell imaging for 2 min.

3) Adjust tothe target laser power.

 

Controlledbacterial cell movement measurement

 

- Materials and Reagents

Protein A magnetic beads (New EnglandBioLabs)

Anti- E. coli antibody (Bioon)

Antibody coating buffer solution - Protein Amagnetic bead binding buffer (New England BioLabs)

LB medium (USB)

Curvette (Sarstedt)

Spectrophotometer

40X bright-field microscope

 

- Protocol

1)Wash the Protein A magnetic bead with antibody coating buffer 3 times.

2)Resuspend the Protein A magnetic bead in antibody coating buffer.

3) Addanti- E. coli antibody and incubate at 4C overnight with gently shaking.

4)Wash away the excess antibody with with antibody coating buffer 3 times.

5)Addition of the E. coli cell to resuspend the magnetic bead and incubate for 2hour room temperature with gently shaking.

6) Themagnetic beads were transferred to a new tube and rinsed with fresh cell medium

7) Thecells are attached with magnetic particles and subjected to movement bymagnetic field.

8) Thenumber of cells captured by fixed antibody-tagged magnetic particles aremonitored by disrupting the antibody through heat or chemical and detectedspectrophotometry or microscopy.

9) The bacterial cell tagged on magneticparticles, which can be observed with bare eyes with a dark brown colour, wasattracted by a fixed magnetic field and the movement rate is calculated bydistance over time until all brown colour particles bind on the wall

of the container.

 

Laserexcitation protocol

General,wavelength

 

-Protocol

1)Grow transformed bacteria BL121(DE)in LB with 0.4 M of NaCl in the presence of antibiotic at 37°C.

2)Collect 500 μl of bacterial sample with OD600 reaches 0.4.

3)Transfer the bacterial sample to cell chamber.

4)Excite bacterial sample by laser confocal scanning microscope (FV1000D, Nikon).

5)Three wavelengths are chose: 405nm, 530 nm and 670 nm.

6)Length for excitation is 2 minutes.

7)Collect 200 μl of excited bacterial sample.

8)Spin sown bacterial sample.

9)Remove supernatant.

10)Lysis bacterial in 90 μl of 2% SDS.

11)Boil at 99°C for 10 minutes.

12)Determine the intracellular Cl^- concentration by MQAE.

 

Laser power

 

-Protocol

1) Grow transformed bacteria BL121(DE) in LB with 0.4 M of NaCl inthe presence of antibiotic at 37°C.

2) Collect 500 μl of bacterialsample with OD600 reaches 0.4.

3) Transfer the bacterial sample tocell chamber.

4) Excite bacterial sample by laserconfocal scanning microscope (FV1000D, Nikon).

5) Wavelength chose 530 nm.

6) Length for excitation is 2minutes.

7) Power of laser: 0-65%; 5% asinterval.

8) Collect 200 μl of excitedbacterial sample.

9) Spin sown bacterial sample.

10) Remove supernatant.

11) Lysis bacterial in 90 μl of 2%SDS.

12) Boil at 99°C for 10 minutes.

13)Determine the intracellular Cl^- concentration by MQAE.

 

Excitation length

 

- Protocol

1) Grow transformed bacteria BL121(DE) in LB with 0.4 M of NaCl inthe presence of antibiotic at 37°C.

2) Collect 500 μl of bacterialsample with OD600 reaches 0.4.

3) Transfer the bacterial sample tocell chamber.

4) Excite bacterial sample by laserconfocal scanning microscope (FV1000D, Nikon).

5) Wavelength chose 530 nm.

6) Length for excitation: 0-600s;30s as interval.

7) Power of laser: 25%.

8) Collect 200 μl of excitedbacterial sample.

9) Spin sown bacterial sample.

10) Remove supernatant.

11) Lysis bacterial in 90 μl of 2%SDS.

12) Boil at 99°C for 10 minutes.

13) Determinethe intracellular Cl^- concentration by MQAE.