User:David Johnston Monje/Protocols

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Housekeeping type protocols

Standard 50 ul PCR recipe for soil DNA

• 37 ul of distilled water
• 5 ul of 10X PCR buffer without Mg
• 3 ul of MgCl
• 1 ul of 25 mM dNTP mix
• 1 ul of 10mM forward primer (usually 10 pmol of primer)
• 1 ul of 10mM reverse primer (usually 10 pmol of primer)
• 0.5 of BSA (bovine serum albumin) to protect against enzyme inhibition
• 0.5 of taq
• 2 ul of template DNA (and no more than 100 ng per reaction)

qPCR Recipe (9 ul total)

• 5 ul SYBR green mix
• 2.75 ul of dH2O
• 0.5 ul of forward primer (10 mM)
• 0.5 ul of reverse primer (10 mM)
• 0.25 ul of BSA
• 1 ul of cDNA template

Ethanol precipitation of DNA

• Mix 95 mL of 100% EtOH with 4 mL of sodium acetate (NaOAc at pH 4.8) and 1 mL dH20.
• Add 2.5 volumes of this to the DNA solution to be precipitated and mix by pipetting or vortexing. For 20 ul of PCR mix add 50 ul of this ethanol:NaOAc:H20 blend for example.
• Place tubes at -20 degrees from 2 hours to overnight.
• Centrifuge tubes at 2 degrees Celcius at maximum speed for 15 minutes.
• Pour off supernatant and place tubes upside down on paper towel for a minute to ensure dryness.
• Add the same volume of cold, 80% EtOH to the DNA pellet and flick once with finger to wash.
• Centrifuge tubes at 2 degrees Celcius at maximum speed for 3 minutes.
• Pour off supernatant and place tubes upside down on paper towel for a minute to ensure dryness.
• Add the same volume of cold, 100% EtOH to the DNA pellet and flick once with finger to wash.
• Centrifuge tubes at 2 degrees Celcius at maximum speed for 3 minutes.
• Pour off supernatant and place tubes upside down on paper towel for 5 minutes to ensure dryness.
• Resuspend the white DNA pellet in your choice of solvent (normally water and 10 ul of it for TRFLP).

20X SSC Buffer (use 0.5X SSC for physical capture TRFLP)

• Dissolve 175.3 g of NaCl and 88.2 g of sodium citrate in 800 ml of H2O. Adjust the pH to 7.0 with a few drops of a 14 N solution of HCl. Adjust the volume to 1 L with H2O and dispense into aliquots. Sterilize by autoclaving. Final concentrations in 20X SSC are 3.0 M NaCl and 0.3 M sodium citrate.

Surface sterilization, sampling and culturing

Phytosphere DNA Extraction

• Leaves or roots were placed in polypropylene tubes and submerged with washing buffer (0.1Mpotassium phosphate buffer, pH 7.0) and sonicated for 7 min in an ultrasonic cleaning bath for 7 min to dislodge bacteria from leaves or roots. This is a method from a previous publication. Similar methods were used for both phylloplane and rhizoplane to ensure comparability of results.

Corn/Teosinte Seed Surface Sterilization Protocol

• Soak 20 seeds in dH2O for 48 hours
• Drain H2O and add Sunlight dish soap + water to the container and seeds and wash for 10 minutes in shaking water bath
• Drain the soapy water and add 2.5% sodium hypochlorite to cover the seeds. Wash for 10 minutes.
• Drain the bleach, and add 2.5% sodium hypchlorite to cover the seeds. Wash 10 minutes.
• Drain the bleach, and add 95% ethanol to cover the seeds. Wash 10 minutes.
• Drain the bleach and do 3 thirty second washes with distilled water.
• To test for sterility, momentarily place the seeds on R2A media and culture that at 25 degrees for 10 days.
• Grind seed in mortar and pestle, adding 1 ml of 2.5 M sodium phosphate buffer for each g of seed dry weight (teosinte takes 2 ml / g)
• Take a 100 ul aliquot of supernatant for culturing on plates, and keep the rest for DNA extraction

Corn/teosinte growth, surface sterilization, and endophyte isolation protocol

• Soak 20 seed in dH2O for 24 hours, then transfer them to substrate of interest, watering as needed.
• Corn and teosinte were grown in growth chamber with 50% rel hum, and 200 photons of light m-2s-1 provided by fluorescent and incandescent bulbs, having a photoperiod of 16-hrs daylight and a 28ºC: 22ºC (light: dark) temperature cycle.
• Observe plant growth for 1 to two weeks and harvest when plants reach the V3 growth stage (5 leaf tips).
• Remove individual plants from pots, gently washing off visible soil in tap water and place the plant in a 100 mL pyrex bottle. Add water and 1 mL of sunlight soap to cover the plant. Wash in shaker for 10 minutes.
• Drain the water, and replace (covering the plant) with 2.5% bleach. Wash for 10 minutes.
• Drain the water/bleach and add 2.5% bleach to cover the plant. Wash for 10 minutes.
• To ensure surface sterility, add 95% ethanol and conduct an additional 10 minute wash to completely surface sterilize the plant.
• Drain the ethanol and do 3 thirty second washes with distilled water.
• To test for sterility, momentarily place the plant roots on R2A media and culture that at 25 degrees for 10 days.
• To isolate endophytes, thoroughly grind the plant (root, shoot, and embryonic seed) in an autoclaved mortar and pestle. Add 1 mL of sterile phosphate buffer to each g of plant fresh weight and reserve 1 mL of homogenate/supernatant for DNA extraction (for TRFLP).
• For culturing dilute the sample by taking 50 ul of juice from the homogenate, and add to 450 ul of sterile buffer in a eppendorf tube.
• Serially dilute the sample by reisolating 50 ul of from the first tube, to a second containing 450 ul of sterile buffer, and then to a third to a final dilution of 1000 X.
• Plate 100 ul of 1000X dilution on LGI, ½ PDA, and R2A (with cyclohexamide) filled petri dishes. Culture at 25 degrees for 10 days.

R2A Agar Medium

• Casein acid hydrolysate 0.5 g/L
• Yeast extract 0.5 g/L
• Proteose peptone 0.5 g/L
• Dextrose 0.5 g/L
• Soluable starch 0.5 g/L
• Dipotassium phosphate 0.5 g/L
• Magnesium sulfate 0.024 g/L
• Sodium pyruvate 0.3 g/L
• Agar 15 g/L (if you want broth leave the agar out)
• Final pH = 7.2

TY (tryptone-yeast) Agar (TYA) for rhizobia

• 5 g/L Tryptone
• 3 g/L Yeast extract
• 1.3 CaCl2-6H2O
• 10 g/L Agar

Carboxymethylcellulose(CMC)Agar Medium

• 0.2% carboxymethylcellulose (CMC) sodium salt and 0.1% triton X-100 were added to R2A media, as per manufacturers instructions, then autoclaved and poured into 150 mm plates
• To visualize cellulase activity, gram's iodine is flooded onto the plate and clear halos measured against the relatively dark background
• Similar to that used in : http://www.springerlink.com/content/q7g54721205r26k3/fulltext.html

Pectin Agar Medium

• 0.2%(w/v) of citrus pectin and 0.1% triton X-100 were added to R2A media, as per manufacturers instructions, then autoclaved and poured into 150 mm plates
• To visualize cellulase activity, gram's iodine is flooded onto the plate and clear halos measured

RNA rich Agar Medium

• 1.5 g of torula yeast RNA was dissolved in 1 mL of 0.1 M PO at pH 8 and filter sterilized with a 0.22 um filter.
• This sterilized RNA was added to 250 mL of autoclaved R2A agar media and poured into 150 mm plates.
• After 5-7 days of microbial growth, plates were flooded with perchloric acid for 5 minutes and scored for clear halo production around colonies.
• Ref: BARC Newsletter Issue No. 24, Founder's Day Special Issue 91. http://barc.gov.in/webpages/letter/2004/200410-13.pdf

Mineral Phosphate Solubilization Agar

• 10 g/L glucose + 0.373 g/L NH4NO3 + 0.41 g/L MgSO4 + 0.295 g/L NaCL + 0.003 FeCL3 + 0.7 g/L Ca3HPO4 + 20 g/L agar
• This was adjusted to pH 6 then autoclaved and poured into 150 mm plates
• After 5 days growth at 25 degrees, clear halos were measured
• Reference : http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T3C-41PNYNT-7&_user=1067211&_rdoc=1&_fmt=&_orig=search&_sort=d&view=c&_acct=C000051237&_version=1&_urlVersion=0&_userid=1067211&md5=1ba0dfc48ab1c3e9a86a73ab85e16484

Auxin production

• R2A agar media was supplimented with L-tryptophan to a final concentration of 5 mM, then autoclaved and poured into 150 mm plates.
• At day 4 the plates were overlaid with nitrocellulose cutouts, and put in a fridge at 4 degrees overnight, allowing bacteria and their metabolites to infiltrate the paper
• On day 5, nitrocellulose membranes were removed and placed on Salkowski reagent (0.01M ferric chloride in 35% perchloric acid) saturated Whatman #2 filter papers for 30 minutes and measured for reddish halos surrounding colonies.
• Ref: Appl Environ Microbiol. 1991 February; 57(2): 535–538. http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pubmed&pubmedid=16348419

Acetoin and diacetyl production

• R2A broth was supplimented with 0.5% glucose, then autoclaved and 1 mL added into each of 96 wells on 96 well plates.
• At day 5 100 ul aliquots of culture were removed and placed in 96 well fluorimeter plates, each of which was then mixed with 100 ul of Barrit's reagents A/B (5 g/L creatine were autoclaved, cooled to 60 degrees, then mixed 3:1 with freshly prepared a-naphthol [75 g/L in 2.5 M sodium hydroxide])
• After 30 minutes, wells were scored for pink,red, or dark red colour relative to negatives which are copper brown or yellow in colour.
• Ref: Journal of Basic Microbiology, Jan 2007, 34(4), Pages 277 - 280 http://www3.interscience.wiley.com/journal/114053778/abstract

A different and simpler method is listed below and was used for screening for transgenic acetoin production in E. coli.

• Using VP broth (Peptone from meat 7.0; D(+)glucose 5.0; sodium phosphate 5.0, pH 5) I cultured Kp342 with pDKS-GFPuv (with or without Kanamycin), DH5alpha with GFPuv (with or without Kanamycin), JM109 (without Kanamycin), and DH5alpha with two different copies of the alsD operon in pDSK-GFPuv (with kanamycin). After 48 hours, I added 0.5 mL Barrit Reagen A (0.5 g 1Napthol in 10 mL EtOH) and 0.5 mL of Barrit Reagent B (40% potatssium hydroxide) to 1 ml of the bacterial cultures and waited 30 minutes to record a reaction. Interestingly, antibiotics seem to inhibit acetoin anabolism from glucose in Kp342 (positive control) at least.

Siderophore production

• Microbes were grown on normal R2A agar media for 5 days at 25 degrees.
• At day 5 the plates were overlaid with 30 mL O-CAS overlay
• Double distilled water was used for preparing the culture media and all glassware was treated with 6 M HCl to remove iron and rinsed with water. (Cox, C.D., 1994. Deferration of laboratory media and assays for ferric and ferrous ions. Methods Enzymol. 235, pp. 315–372.Cox, 1994).
• 1 liter of O-CAS overlay was made by mixing Chrome azurol S (CAS) 60.5 mg, hexadecyltrimetyl ammonium bromide (HDTMA) 72.9 mg, finely crused/powdered Piperazine-1,4-bis(2-ethanesulfonic acid) (PIPES) 30.24 g, 1 mM FeCl3 · 6H2O in 10 mM HCl 10 mL and 1% Agarose. Again, the PIPES has to be finely powdered and mixed gently with the O-CAS by stirring (not shaking) to avoid producing bubbles. The 1% agarose is added to pre-warmed O-CAS just prior to pouring the overlay in a proportion of 1:3. After 15 minutes, colour change is scored (purple or orange halos).
• After 15 minutes, a change in color will be observed in the overlaid medium, exclusively surrounding producer microorganisms, from blue to purple (as described in the traditional CAS assay for siderophores of the catechol type) or from blue to orange (as reported for microorganisms that produce hydroxamates).
• Ref: Journal of Microbiological Methods Volume 70, Issue 1, July 2007, Pages 127-131
• http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T30-4NGRRNR-1&_user=1067211&_rdoc=1&_fmt=&_orig=search&_sort=d&view=c&_acct=C000051237&_version=1&_urlVersion=0&_userid=1067211&md5=e3666005a65ed298d3d5733749ff485e

ACC Deaminase Activity

• Double distilled water was used and all glassware was treated with 6M HCl to clean it before media preparation
• LGI media (nitrogen free) was made and 1ul/mL of 2M ACC in H20 was included as a nitrogen source in 1 mL aliquots on a 96 well plate.
• 96 well plates were then sealed and incubated at 25 degrees with shaking for 10 days.
• Non-diazotrophic bacteria that grow in this assay have a noticibly higher OD600 after 10 days growth using ACC as a nitrogen source and thus posses an ACC deaminase activity

Growth on nitrogen free media : LGI

• Double distilled water was used and all glassware was treated with 6M HCl to clean it before media preparation
• 50 g Sucrose
• 0.01g FeCl3-6H2O
• 0.8g K3PO4
• 0.2g MgSO4-7H20
• 0.2 g CaCl2
• 0.002g Na2MoO4-2H2O
• pH 7.5 --> then autoclave and put 1 mL aliquots into a 96 well plate and incubate at 25 degrees with gentle shaking for 10 days, before taking OD600 plate reading to estimate growth.

Potato Nodal Cutting Media

• Murashige Skoog Basal Salts with Minimal Organics (Sigma - M6899) 4.4 g/L
• Sucrose 15 g/L
• Agar 7.5 g/L
• pH = 6
• Autoclave and put 10 mL in each 22x150 mm tissue culture tube with a potato node and incubate for a month in a chamber with 50% humidity, 100 umol photons m2s1 and 16 hours daylight and 24 degree Celcius day/16 degree Celcius night
• Reference: http://rparticle.web-p.cisti.nrc.ca/rparticle/AbstractTemplateServlet?calyLang=eng&journal=cjm&volume=43&year=&issue=9&msno=m97-117

NBY AGAR rich media for growing most plant pathogenic bacteria 1000mL 800mL 500mL 100mL Nutrient Broth 8.0g 6.4g 4.0g 0.8g Yeast Extract 2.0g 1.6g 1.0g 0.2g K2PHO4 2.0g 1.6g 1.0g 0.2g (or K2HPO4 3H2O) (2.62) (2.10) (1.31) (0.262) KH2PO4 0.5g 0.4g 0.25g 0.05g Bacto-Agar 15.0g 12.0g 7.5g 1.5g H2O 950mL 760mL 475mL 95mL Autoclave, cool to 50 degrees C and add 1.0 M MgSO4 1.0mL 0.8mL 0.5mL 100uL 10 % Glucose 50mL 40mL 25mL 5mL

LB (LURIA-BERTANI) AGAR 1000mL 800mL 500mL 100mL Tryptone 10.0g 8.0g 5.0g 1.0g NaCl 5.0g 4.0g 2.5g 0.5g Yeast Extract 5.0g 4.0g 2.5g 0.5g Agar 15.0g 12.0g 7.5g 1.5g H2O 1000mL 800mL 500mL 100mL pH to 7.4-7.5 before adding agar, then autoclave.

KING'S B AGAR 1000mL 500mL 250mL 100mL Tryptone 10.0g 5.0g 2.5g 1.0g Protease Peptone #3 10.0g 5.0g 2.5g 1.0g Glycerol 10.0mL 5.0mL 2.5mL 1.0mL Agar 15.0g 7.5g 3.75g 1.5g Check pH before adding agar, adjust to pH 7.2-7.3. Autoclave, then cool to 50 degrees C and add: K2HPO4 (0.5M) 17.2mL 8.6mL 4.3mL 1.7mL MgSO4 7 H2O(1.0M) 6.1mL 3.05mL 1.5mL 0.6mL (Modification of original recipe by King, Ward and Raney, 1954)

CKTM Agar for bacterial spot (Xanthomonas campestris pv. vesicatoria) Per Liter Soy Peptone 2.0 g Tryptone 2.0 g Glucose 1.0g L-glutamine 6.0 g Ammonium Phosphate (NH4)2HPO4 0.8g KH2PO4 1.0g Agar 12.0 g Make up to 980 ml with water Autoclave together for 20-25 minutes in a 2 L flask with a stir bar Tween 80 10 ml Autoclave separately and add to the above mixture as soon as they come out of the autoclave with stirring. The mixture will be cloudy until it cools to 50C Cool to 50C and then add to the mixture above (with continual stirring) MgS04 (1 M) 1.63 ml sterilize by autoclaving CaCl2 (1 M) 1.8 ml sterilize by autoclaving cycloheximide (75mg/ml) 1.33 ml Make up in 70% ethanol bacitracin (50 mg/ml) 2.0 ml Make up in water and filter sterilize neomycin sulfate (20 mg/ml) 0.5 ml Make up in water and filter sterilize cephalexin (25 mg/ml) 2.6 ml Make up in 70% ethanol tobramycin (4 mg/ml) 0.1 ml Make up in water and filter sterilize Note: all antibiotics are stored at -20C and melted if necessary before use. Ethanol will sterilize compounds made up in it so it is not necessary to filter sterilize them. Note: cephalexin will NOT dissolve in ethanol and remains as a suspension. Make sure that it is vortexed immediately before use as the cephalexin will settle out of suspension. Check when pouring plates that you are pouring them thick enough. It is easy to pour them too thin - I think the detergent (Tween 80) makes the agar more slippery and it covers the bottom of the plate more quickly than media without a detergent. Ref: Sijam/Chang/Gitaitis in Phytopathology (1991) 81: 831-34

Vogel-Bonner Tartrate Medium (VBTar) for bacterial speck (Pseudomonas syringae pv. tomato) I. Agar base Purified (or Noble) agar 18.0 g water 940ml Autoclave in a 2 liter flask with a stir bar for 25 minutes and cool in a water bath to 50-55C II. To the cooled agar add: 40X VB salts 25 ml 10% Tartaric Acid 30 ml 1 M MgSO4 0.8 ml 1000X FeCl3.6H2O 1.0 ml 2N NaOH about 9-11 ml Make sure the VB salts and the 10% tartaric acid are warmed in the water bath for about 30 minutes before you add to the agar. Otherwise such a large volume of room temperature solutions will cause the agar to gel and you will have lumps and non-homogenous media. Put the cooled (to 55C) agar on a stirrer and add the 40X VB salts, the tartaric acid, the MgSO4 and the FeCl3 to the agar while stirring. Add about 10 ml of the 2N NaOH to the mixture and stir well. Remove a small amount of the liquid media with a sterile pasteur pipette and spot it on a pH indicator strip. The pH should be about 7 -7.4 If the pH is below 7 add more NaOH until the pH is correct.

40 X VB Salts K2HPO4 160 g NaH2PO4.H20 37.08 g NH40H 16. 2ml H20 to 400 ml Put about 300 ml water in a beaker, add the salts and dissolve. Check pH and add conc. HCl to adjust to pH 7.5. Add water to make a total of 400 ml. Autoclave.

10 % Tartaric Acid D (-) Tartaric Acid 10 g H20 to 100 ml Filter Sterilize by passing the solution through a sterile 0.2 or 0.45  filter into a pre-sterilized bottle. DO NOT AUTOCLAVE - the heat of autoclaving will break down the tartaric acid 1000 X Ferric Chloride FeCl3.6H20 250 mg H20 100 ml . Filter Sterilize. Eventually the ferric chloride will start to precipitate out of solution. When it does, make a fresh batch.

CNS medium for bacterial canker (Clavibacter michiganensis subsp. michiganensis) (Gross, D. and A.K. Vidaver. 1978. A selective medium for isolation of Corynebacterium nebraskense from soil and plant parts. Phytopathology 69:82-87)(Also in Laboratory Guide for Identification of Plant Pathogenic Bacteria, 3rd Edition. 2001. Editors: N.W. Schaad, J.B. Jones and W. Chun. APS Press) per liter: Nutrient Broth 8.0 g Yeast Extract 2.0 g K2HPO4 2.0 g KH2PO4 0.5 g Lithium chloride 5.0 g Agar 15.0 g Autoclave 20-25 min in 2-L flask with a stir bar. Cool to 50 C and add with continual stirring: Glucose (sterile 10% solution) 50 ml MgSO47 H2O (sterile 1 M sol.) 1 ml Cycloheximide (75 mg/ml) 0.53 ml (made up in 70% ethanol) Nalidixic acid (5 mg/ml) 5.0 ml (made up in 0.1 N NaOH) Polymyxin B (10 mg/ml) 3.2 ml (made up in water and filter-sterilized) Pour plates. Note: Cycloheximide, nalidixic acid and polymyxin B are stored at 4 C Note: Original recipe had Daconil 2787-F but we did not use it.

Bacterial Transformation

Preparation of electrocompetent E. coli

• Innoculate 1 litre of LB broth (adapt with R2A for other bacteria) with 1/100 of a fresh overnight culture.
• Grow at 37 degrees with shaking until it reaches OD600 = 0.5 - 1.0
• Harvest the cells by chilling for 15 minutes on ice, then centrifuge in cold rotor at 4000 g for 15 minutes.
• Remove the supernatant and resuspend in 1 L of 4 degree water
• Centrifuge as in 3 above, and remove supernatant again.
• Resuspend in 0.5 L cold water and centrifuge again
• Remove supernatant and resuspend in cold 20 mL of 10% glycerol.
• Centrifuge and remove the supernatant again. Resuspend the pellet in 3 mL of 10% glycerol.
• Make 50 ul aliquots and use fresh for electroporation or freeze at -70 for later.

Electrotransformation of E. coli

• Gently thaw cells on ice.
• In the same tube cells come in, mix in 10 ul of concentrated plasmid and let sit on ice for 1 minute. IMPORTANT: If using a ligation mixture for transformation, first inactivate the ligase with 65 degrees for 10 minutes, followed by two-fold dilution in dH2O.
• Set the electroporator to the appropriate E. coli setting. For the Biorad electroporator with 0.22 um cuvettes, use the EcoRI setting.
• Place the cells in a cold electroporation cuvette and shake to the bottom. Slide it into the machine.
• Press the button to pulse electricity into the cells. The time constant should be 4-5 seconds and electric popping should not happen. Field strength should be 12.5 kV.
• Immediately remove cuvette an quickly add 1 mL of cold SOC medium (or R2A plus 0.25% glucose) , mixing and resuspending cells using pasteur pipette. (time here is very important)
• Remove the cells to a 17x100 mm polhypropelene tube and incubate at 27 for 1 hour with shaking.
• Plate the cells on selective medium