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Contents 1 Protocol 1: LB Broth and Agar 1.1 LB Medium (Luria-Bertani) 1.2 Agar Plates 1.3 Antibiotics 2 Protocol 2: Transforming BioBricks 3 Protocol 3: Minimal Media and Trace Elements 3.1 Trace Elements 3.2 M9 3.3 Tips 4 Protocol 4: Primer Design 5 WE NEED A PRIMER DESIGN PROTOCOL HERE 6 Protocol 5: Qiagen Minipreps 6.1 Procedure 7 Protocol 6: Induction of Luciferase by Toluene and its Derivatives 8 Protocol 7: Restriction Digests 8.1 Roche 8.2 Promega 9 Protocol 8: MoBio PowersoilTM DNA Purification Kit 10 WE NEED A POWERSOIL KIT PROTOCOL HERE 11 Protocol 9: PCR 11.1 2x ReddyMix PCR Master Mix 11.2 Touch 2 11.3 Gradient Program 11.4 KOD Polymerase 11.5 KOD Program 11.6 KOD XL Polymerase 12 Protocol 10: Miller Assay 13 Protocol 11: Gel Extraction 14 Protocol 12: TOPO Cloning Reaction 15 Protocol 13: One Shot Chemical Transformation Protocol for Competent E. coli

Protocol 1: LB Broth and Agar

LB Medium (Luria-Bertani)

Per litre: To 950 ml ddH2O add:

• Tryptone 10 g
• Yeast extract 5 g
• NaCl 10 g

1. Shake until all solutes dissolved.
2. Adjust pH to 7.0 with 5 M NaOH (~0.2 ml).
3. Adjust volume to 1 litre with ddH2O.

Agar Plates

1. Add agar to 15 g/litre into Schott bottle (only fill to 3/4 full).
2. Add appropriate volume of LB and shake to distribute agar.
3. Loosen caps and sterilise by autoclaving. Remove the bottle from the autoclave using heat-proof gloves, tighten the cap, shake to assure the agar is uniformly suspended, and place in a preheated 60ºC water bath. Allow the agar to cool to 60ºC. (Can reheat agar in microwave.)
4. Add appropriate antibiotic solution and shake to distribute uniformly.
5. Pour approximately 3-4 mm of agar into each plate (about 25 ml).
6. Label the edge of the plate with the appropriate colour code for the antibiotic with a magic marker down the side of the plate stack.
7. Stack plates at back to the sterile hood to cool and evaporate the excess water for about 30 min-1 h.
8. After cooling package the plates into the plastic bag the plates came in, seal with lab tape, label with plate agar and antibiotic type, initials, and the date and place in the 4ºC refrigerator.
9. Refrigerated plates are good for several months. Eventually, antibiotic stability or mould growth limits lifetime. Any plates seen with mould growth should be disposed of as biohazard waste without opening to avoid the spreading of the mould spores.

Antibiotics

Antibiotic	Final Concentration	Stock Concentration	Solvent Solution	Colour Code
No antibiotic	_	_	_	Blue
Carbenicillin	50 µg/ml	50 mg/ml	60 % Ethanol	Red
Kanamycin	50 µg/ml	50 mg/ml	H2O	Green
Chloramphenicol	170 µg/ml	34 mg/ml	Ethanol	Black
Tetracycline	50 µg/ml	5 mg/ml	Ethanol	Purple
Streptomycin	50 µg/ml	10 mg/ml	H20	Yellow

Stock solution of antibiotics are stored at -20ºC at the stock solution concentration. Different antibiotics and additives are dissolved in different solvents. Altought carbenicillin is soluble in water, it is convenient to dissolve it in 60% ethanol, which prevents the solution from freezing. Antibiotics dissolved in water are sterilized by filter sterilization using disposible 0.22 µm filters. Antibiotics sterilised in ethanol need not be sterilised.

N.B. Use media without magnesium ions (eg LB) for selection on tetracycline.

Protocol 2: Transforming BioBricks

1. Split the competent E. coli cells (~75 µl) in two. (DB3.1 cells required if plasmid encodes ccdB; otherwise use TOP10)
2. Competent cells (~33 µl) into eppendorfs on ice.
3. Add 1µl of plasmid DNA.
4. Incubate on ice for at least 5 mins.
5. Heat shock +42ºC for 45 secs.
6. Back on ice for 2 mins to reduce damage.
7. Add 1ml of soc media.
8. Incubate +37ºC in a shaker for 1 hour.
9. Spread the reaction on kan/carb LB plates.
10. Grow +37ºC overnight.

Protocol 3: Minimal Media and Trace Elements

Trace Elements

1. In 800 ml dH2O dissolve 5 g Na2 EDTA and convert to pH7.
2. Add the following in order, correcting to pH7 after each.
   • FeCl3 (.H2O) 0.5 (0.83 g)
   • ZnCl2 0.05 g
   • CuCl2 0.01 g
   • CoCl2.6H20 0.01 g
   • H3BO3 0.01 g
   • MnCl2.6H2O (.4H2O) 1.6 g (1.35 g)
3. Make up to 1 litre, autoclave and store at 4°C.

M9

1. Obtain M9 (or M9- for 15N labelling) from kitchen. Per litre, kitchen adds
   • 6 g Na2HPO4
   • 3 g KH2PO4
   • 0.5 g NaCl
2. The add
   • 1 ml 1M MgSO4
   • 200 µl 1M CaCl2
   • 1 ml Thiamine (40mg ml-1 stock)
   • 10 ml Trace Elements
3. Also add as necessary
   • 15 ml (13C-) Glucose (20% stock) (gives 0.3% final)
   • 1 g (15) NH4Cl (if using M9-)

Tips

1. Grow from fresh transformation into rich media overnight, and use that to sub-culture labelling media at 1:50 for induction.
2. Instead of glucose, succinate may be used to prevent inhibition reactions.

Protocol 4: Primer Design

WE NEED A PRIMER DESIGN PROTOCOL HERE

Protocol 5: Qiagen Minipreps

This protocol is designed for purification of up to 20 μg of high-copy plasmid DNA from 1–5 ml overnight cultures of E. coli in LB (Luria-Bertani) medium. Note: All protocol steps should be carried out at room temperature.

Procedure

1. Resuspend pelleted bacterial cells in 250 µl Buffer P1 (kept at 4°C) and transfer to a microcentrifuge tube. Ensure that RNase A has been added to Buffer P1. No cell clumps should be visible after resuspension of the pellet.
2. Add 250 μl Buffer P2 and gently invert the tube 4–6 times to mix. Mix gently by inverting the tube. Do not vortex, as this will result in shearing of genomic DNA. If necessary, continue inverting the tube until the solution becomes viscous and slightly clear. Do not allow the lysis reaction to proceed for more than 5 min.
3. Add 350 μl Buffer N3 and invert the tube immediately but gently 4–6 times. To avoid localized precipitation, mix the solution gently but thoroughly, immediately after addition of Buffer N3. The solution should become cloudy.
4. Centrifuge for 10 min at 13,000 rpm (~17,900 x g) in a table-top microcentrifuge. A compact white pellet will form.
5. Apply the supernatants from step 4 to the QIAprep spin column by decanting or pipetting.
6. Centrifuge for 30–60 s. Discard the flow-through. Spinning for 60 seconds produces good results.
7. (Optional): Wash the QIAprep spin column by adding 0.5 ml Buffer PB and centrifuging for 30–60 s. Discard the flow-through. This step is necessary to remove trace nuclease activity when using endA+ strains such as the JM series, HB101 and its derivatives, or any wild-type strain, which have high levels of nuclease activity or high carbohydrate content. Host strains such as XL-1 Blue and DH5α do not require this additional wash step. Although they call this step optional, it does not really hurt your yield and you may think you are working with an endA- strain when in reality you are not. Again for this step, spinning for 60 seconds produces good results.
8. Wash QIAprep spin column by adding 0.75 ml Buffer PE and centrifuging for 30–60 s. Spinning for 60 seconds produces good results.
9. Discard the flow-through, and centrifuge for an additional 1 min to remove residual wash buffer. IMPORTANT: Residual wash buffer will not be completely removed unless the flow-through is discarded before this additional centrifugation. Residual ethanol from Buffer PE may inhibit subsequent enzymatic reactions. They are right about this.
10. Place the QIAprep column in a clean 1.5 ml microcentrifuge tube. To elute DNA, add 50 μl Buffer EB (10 mM Tris·Cl, pH 8.5) or water to the center of each QIAprep spin column, let stand for 1 min, and centrifuge for 1 min. If you are concerned about the concentration of the DNA, you can alternatively add 30 μl water to the center of the column, incubate at room temperature on the bench for 5 mins and then centrifuge for 1 min. This will increase the concentration of DNA in your final sample which can be useful in some cases. You should elute in water rather than the Buffer EB they recommend if you plan to sequence your sample. Even if you are not sequencing, it may be beneficial to elute in water. For instance, if you elute in buffer EB and you are using this DNA in a restriction digest, then the additional salts in your sample can affect the salt content of your digest. This may matter with some finicky enzymes.

Protocol 6: Induction of Luciferase by Toluene and its Derivatives

(adapted from Willardson et al., 1998) See References

1. Grow single colonies of DH5α cells containing pGLTUR in 10 ml LB medium containing 100 µM carbenicillin at 37ºC until OD600 is between 0.2 and 1.0.
2. Dilute to an OD600 of 0.2 with LB medium.
3. Induce luciferase transcription by mixing 0.9 ml of the diluted culture with 0.9 ml of LB medium containing increasing amounts of toluene or its derivative compounds.
4. Incubate in 2.0 ml glass vials (sealed with Teflon septa to avoid loss of the volatile organic compound) at 37ºC for 30 min and then cool on ice for 10 min.
5. Measure luciferase activity
6. 75 µl of the induced culture is lysed by addition of 25 µl of 4x lysis buffer (100 mM Tris.HCl [pH 7.8], 32 mM NaH2PO4, 8mM dithiothreitol, 8 mM CDTA, 4% (vol/vol) Triton-X 100, and 200 µg of polymyxin B sulphate per ml).
7. 25 µl of the lysed cell solution was added to 25 µl of a combined 4x concentrate of luciferase substrates A and B (Analytical Luminescence Laboratories) to give a final 2x concentration of each substrate.
8. Luminesence read immediately after substrate addition for 45 s in a Turner TD-20e luminometer.

Protocol 7: Restriction Digests

Roche

• 4ul Miniprep DNA
• 1ul Enzyme 1
• (1ul Enzyme 2 if double digest)
• 2ul Buffer
• 12ul H2O (so to make up to 20ul total)

Add enzymes last!

Promega

• 2ul 10 x Diluted BSA stock
• 4ul Miniprep DNA
• 1ul Enzyme 1
• (1ul Enzyme 2 if double digest)
• 2ul Buffer
• 10ul H2O (so to make up to 20ul total)

Add enzymes last!

Protocol 8: MoBio PowersoilTM DNA Purification Kit

WE NEED A POWERSOIL KIT PROTOCOL HERE

Protocol 9: PCR

2x ReddyMix PCR Master Mix

• From Thermo Scientific
• Contains:
  1. The Thermoprime Plus DNA Polymerase
  2. dNTPs
  3. Reaction Buffer
  4. Magnesium Chloride
  5. Pink Dye

Touch 2

1. 94ºC 15:00
2. 94ºC 00:20
3. 65ºC 00:30
4. 72ºC 01:30
5. Go to step 2. once.
6. 94ºC 00:20
7. 64ºC 00:30
8. 72ºC 01:30
9. Go to step 6. once.
10. 94ºC 00:20
11. 63ºC 00:30
12. 72ºC 01:30
13. Go to step 10. once.
14. 94ºC 00:20
15. 62ºC 00:30
16. 72ºC 01:30
17. Go to step 14. once.
18. etc.

This PCR program will decrease the melting temperature by 1ºC every two cycles until Tm reaches 55ºC.

Gradient Program

1. 94ºC 10:00
2. 94ºC 00:20
3. Annealing temperature 00:30
4. 72ºC 01:30
5. Go to step 2. for 29 times.
6. 72ºC 05:00
7. 4ºC for ever

This PCR program will use different Tm temperatures varying from 70ºC to 55ºC across the plate creating a temperature gradient from one side to the other.

KOD Polymerase

KOD polymerase combines high fidelity, fast extension speed and outstanding processivity (DNA pol + 3' exonuclease activity) with high yield and specificity of antibody based Hot Start technology which inhibits enzyme activity during PCR assemby and initial denaturation step. Generates blunt ended PCR fragments suitable for cloning.

• Reaction setup:
  • 5ul 10 x reaction buffer
  • 3ul MgSO4
  • 5ul dNTPs
  • 32ul dH2O
  • 1.5ul forward primer
  • 1.5ul reverse primer
  • 1ul DNA
  • 1ul KOD Hotstart Polymerase

KOD Program

1. Polymerase activation 95C for 2 mins
2. Denature 95ºC for 20 secs
3. Annealing 63ºC for 10 secs
4. Elongation 70ºC for 1 min
5. Repeat to step 2 for 25 times
6. 70ºC for 2 mins
7. 4ºC forever

KOD XL Polymerase

This is a mutant form of KOD that lacks 3´→ 5´ exonuclease activity. This enzyme is used to amplify long targets with high processivity high accuracy.

Protocol 10: Miller Assay

This Miller assay is adapted from the protocol described on the open wet ware.

E. coli culture grown overnight in LB at 37ºC. The following morning the culture had 5 ml aliquots poured into 15 ml tubes. A range of concentrations of salicylate were added. The induced cultures were left to grow for a further 3 hours at 28ºC. While cultures were growing, aliquots of 80 μl permeabilization solution were added to 1.5 ml Eppendorfs. After 3 hours, 1 ml of culture from each tube was added to a cuvette, placed in a spectrophotometer and absorbance at 600nm (A600) was recorded. A 1 ml pure LB blank reference had been recorded previously. A 20 μl sample from each culture and was added to permeabilization solution aliquots. After this step the reaction was stable for a few hours.

Samples along with the substrate solution were moved to a 30ºC water bath for 30 minutes. Each tube had 600 μl substrate solution added and the time of addition was recorded. The reaction was allowed to continue until a desired colour development had been achieved, after which 700 μl of stop solution was added, the time noted and the tubes mixed well. After the last sample was stopped, the tubes were transferred to a microcemtrifuge and spun for 5-10 minutes at the top speed. The tubes were then carefully removed from the centrifuge and the solution at the very top of the tubes transferred to cuvettes. The A420 was then recorded, with an expected range between 0.05 and 1.

With this modified assay, Miller units are calculated thus:

____________________1000 x (A420)__________________
((A600of culture) x (volume 0.02 ml) x (reaction time in mins))

Recipes for solutions (500 ml)

1. Permeabilization solution
   • 7.098 g 100 mM Na2HPO4
   • 0.746 g 20 mM KCl
   • 0.246 g 2 mM MgSO4
   • 4.0 ml 0.8 mg/ml CTAB
   • 0.200 g 0.4 mg/ml sodium deoxycholate
   • 2.7 ml 5.4 μl/ml β-mercaptoethanol
2. Substrate solution
   • 4.258 g 60 mM Na2HPO4
   • 3.120 g 40 mM NaH2PO4
   • 0.500 g 1 mg/ml ONPG (ONPG to be added just before use)
   • 1.35 ml 2.7 μl/ml β-mercaptoethanol
3. Stop solution
   • 53.0 g 1 M Na2CO3

NB! When using β-mercaptoethanol it is advisable to add this to an aliquoted amount of permeabilization and substrate solutions, when they are to be used, as β-mercaptoethanol will degrade after a few days. This will allow for keeping a stock of both solutions in the lab.

Protocol 11: Gel Extraction

QIAGEN QIAquick Gel Extraction Microcentrifuge Protocol

1. Excise DNA fragment from agarose.
2. Weigh gel slice, add 3 volumes of Buffer QG to 1 volume of gel (100ug ~ 100ul).
3. Incubate at 50C for 10 min to dissolve gel completely. Vortex every 2-3 min during the incubation.
4. After the gel slice has dissolved completely, check that the colour is yellow. If orange or violet add 10 ul of 3M sodium acetate pH5.0 and mix. Will turn yellow.
5. Add 1 gel volume isopropanol.
6. QIAquick spin column in 2ml collection tube.
7. To bind DNA, apply the sample to the QIAquick column and centrifuge for 1 min. Discard flow-through and place the QIAquick column back in the same tube.
8. Recommended: Add 0.5ml Buffer QG to QIAquick column and centrifuge for 1 min. Discard flow-through and place the QIAquick column back into the same tube.
9. To wash, add 0.75ml of Buffer PE to QIAqucik column and centrifuge for 1 min. Discard flow-through.
10. entrifuge for 1 min at 13000rpm in 2ml collection tube.
11. Place column in a clean 1.5 microcentrifuge tube.
12. To elute DNA, add 50ul Buffer EB or water (60-70C) to the centre of the QIAquick membrane and centrifuge the column for 1 min. Alternatively, for increased DNA concentration add 30ul elution buffer to the centre of the QIAquick membrane, let the column stand for 1 min, and then centrifuge for 1 min.
13. If the purified DNA is to be analysed on a gel, add 1 volume of loading dye to 5 volumes of purified DNA.

Protocol 12: TOPO Cloning Reaction

From Invitrogen pENTR Directional TOPO Cloning Kits Instruction Manual: Version D, June 2004

For optimal results be sure to use a 0.5:1 to 2:1 molarratio of PCR product:TOPO vector in your TOPO Cloning Reaction.
Note: The blue colour of the TOPO vector solution is normal and it is used to visulaise the solution.

• 0.5 to 4ul Fresh PCR product
• 1ul Salt solution
• xul Sterile Water (so total volume of mix is 6ul)
• 1ul TOPO vector

1. Mix reaction gently and incubate for 5 mins at room temperature (22-23C).
   • Note: For most applicetions, 5 mins will yield a sufficient number of colonies for analysis. Depending on needs (<1kb usually 30 secs, >1kb or if TOPO cloning a pool of PCR products, increasing the reaction time may yield more colonies.
2. Place the reaction on ice and proceed to One Shot Chemical Transformation Protocol for Competent E. coli .
   • Note: You may store the TOPO Cloning Reaction at -20C overnight.

Protocol 13: One Shot Chemical Transformation Protocol for Competent E. coli

From Invitrogen pENTR Directional TOPO Cloning Kits Instruction Manual: Version D, June 2004

Use the following protocol to transform OneSHot TOP10 or Mach I-TI chemically competent E. coli.

1. Add 2ul of the TOPO cloning reaction from TOPO Cloning Reaction, Step 2, into a vial of chemically competent E. coli and mix gently.
2. Incubate on ice for 5 to 30 mins.
3. Heat-shock cells for 30 secs at 42C without shaking.
4. Immediately transfer the tubes to ice.
5. Add 250ul of room temperature LB.
6. Cap the tube tightly and shake horizontally (200rpm) at 37C for 1 hour.
7. Spread 50-200ul from each transformation on a prewarmed selective plate and incubate o/n at 37C. Spread 2 diff volumes to ensure that at least one plate will ahve well spaced colonies.
8. An efficient TOPO Cloning Reaction may produce several hundred colonies. Pick 5-10 for analysis.