User:DrJones1935/12 July 2012

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(Created page with "====I: Check WT B. subtilis Liquid Culture for Growth==== *RESULTS: Culture appears uniformly cloudy consistent with overnight growth. The glycerol stocks are viable.")
(→I: Check WT B. subtilis Liquid Culture for Growth)
 
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====I: Check WT B. subtilis Liquid Culture for Growth====
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====I. Check Plates and Cultures for Growth====
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*RESULTS: Culture appears uniformly cloudy consistent with overnight growth. The glycerol stocks are viable.
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''RESULTS (~09:00)'':
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*No growth on plates (incubator or benchtop) or liquid cultures.
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:*Shaking incubator somehow was at a temperature above 40 <sup>o</sup>C when I arrived this morning, which may have killed cultures.
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:*Poor plating technique may be the cause of the ''B. subtilis'' failure, but the ADP1''ΔmutS'' strain still has not grown on the <span style="color:#00CC00">Kanamycin</span> plate. <strike>I will replate everything and do a parallel streak on LB for the ADP1''ΔmutS'' strain to see if it is really <span style="color:#00CC00">Kan</span><sup>r</sup>.</strike>
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''UPDATE: After changing the temperature on the shaking incubator to once again stabilize at 34 <sup>o</sup>C and allowing the cultures to incubate for the day (~5-6 hours), the pBAV1K-T5-gfp cultures grew turbid. I believe that the 40 <sup>o</sup>C temperature arrested cell growth and that now that the incubator is back at a reasonable temperature, the cultures should grow fine. Considering the plates were getting old and David just streaked out new plasmid strains today, I will wait to make another liquid culture/miniprep until tomorrow.''
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''Also, it is possible that the <span style="color:#00CC00">Kan</span> plates that I was using were too concentrated. I thought they were 10 ug/mL but they were actually 50 ug/mL. I have made more dilute plates and will try plating again tomorrow.'' ~ [[User:Srk3|Srk3]] ([[User talk:Srk3|talk]]) 16:07, 12 July 2012 (EDT)
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====II. Pour plates, selection plates for <span style="color:#00CC00">Kan</span> (12.5 ug/mL)====
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*Weigh out <strike>12.5</strike> 12.50291 g of LB (Miller) medium powder
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*Add dH<sub>2</sub>O water to 350 mL and allow solids to dissolve fully
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*Bring water to 0.5 L and add 0.5 mL of 1 N NaOH solution, mix
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*Distribute solution evenly into 2x 500-mL E. flasks (250 mL in each) and add 1.5% w/v Bacto-agar (<strike>3.75</strike> 3.75025, 3.74982 g/250 mL) to each flask, mix to dissolve solids
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*Loosely cover each flask with foil
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*Autoclave (Liquid cycle, ~50 min)
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*Allow flasks to cool to <60 <sup>o</sup>C and add the appropriate antibiotic to each solution of LB-agar according to the following dilutions:
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{| class="wikitable" border="1"
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|-
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! Antibiotic
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! Volume added stock
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! Final Concentration
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|-
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| <span style="color:#00CC00">Kan</span>
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| 62.5 uL (50 mg/mL)
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| 12.5 ug/mL
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|-
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| LB alone
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| --
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| --
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|}
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*Pour 9-10 plates (~25 mL LB-agar soln. per plate) of each
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*Allow to cool overnight before use
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*Store set plates in sleeve at 4 <sup>o</sup>C for up to 6 months
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====III. Re-streak ''B. subtilis'' Plates====
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*Using sterilized inoculating loop, scrape top layer of ice of glycerol stock and streak cells (trying to thin better for colonies) onto appropriate plate according to the following table:
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{| class="wikitable" border="1"
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|-
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! Strain
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! Plate
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|-
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| ''B. subtilis'' 168 (IA1)
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| LB
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|-
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| ''B. subtilis'' PY79 (IA747)
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| LB
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|-
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| ''B. subtilis'' 1A822
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| LB + <span style="color:#660066">Spc</span>
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|}
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*Place ''B. subtilis'' plates in the static incubator (~34-35 <sup>o</sup>C) overnight

Latest revision as of 23:18, 3 October 2012

I. Check Plates and Cultures for Growth

RESULTS (~09:00):

  • No growth on plates (incubator or benchtop) or liquid cultures.
  • Shaking incubator somehow was at a temperature above 40 oC when I arrived this morning, which may have killed cultures.
  • Poor plating technique may be the cause of the B. subtilis failure, but the ADP1ΔmutS strain still has not grown on the Kanamycin plate. I will replate everything and do a parallel streak on LB for the ADP1ΔmutS strain to see if it is really Kanr.

UPDATE: After changing the temperature on the shaking incubator to once again stabilize at 34 oC and allowing the cultures to incubate for the day (~5-6 hours), the pBAV1K-T5-gfp cultures grew turbid. I believe that the 40 oC temperature arrested cell growth and that now that the incubator is back at a reasonable temperature, the cultures should grow fine. Considering the plates were getting old and David just streaked out new plasmid strains today, I will wait to make another liquid culture/miniprep until tomorrow.

Also, it is possible that the Kan plates that I was using were too concentrated. I thought they were 10 ug/mL but they were actually 50 ug/mL. I have made more dilute plates and will try plating again tomorrow. ~ Srk3 (talk) 16:07, 12 July 2012 (EDT)


II. Pour plates, selection plates for Kan (12.5 ug/mL)

  • Weigh out 12.5 12.50291 g of LB (Miller) medium powder
  • Add dH2O water to 350 mL and allow solids to dissolve fully
  • Bring water to 0.5 L and add 0.5 mL of 1 N NaOH solution, mix
  • Distribute solution evenly into 2x 500-mL E. flasks (250 mL in each) and add 1.5% w/v Bacto-agar (3.75 3.75025, 3.74982 g/250 mL) to each flask, mix to dissolve solids
  • Loosely cover each flask with foil
  • Autoclave (Liquid cycle, ~50 min)
  • Allow flasks to cool to <60 oC and add the appropriate antibiotic to each solution of LB-agar according to the following dilutions:
Antibiotic Volume added stock Final Concentration
Kan 62.5 uL (50 mg/mL) 12.5 ug/mL
LB alone -- --
  • Pour 9-10 plates (~25 mL LB-agar soln. per plate) of each
  • Allow to cool overnight before use
  • Store set plates in sleeve at 4 oC for up to 6 months


III. Re-streak B. subtilis Plates

  • Using sterilized inoculating loop, scrape top layer of ice of glycerol stock and streak cells (trying to thin better for colonies) onto appropriate plate according to the following table:
Strain Plate
B. subtilis 168 (IA1) LB
B. subtilis PY79 (IA747) LB
B. subtilis 1A822 LB + Spc
  • Place B. subtilis plates in the static incubator (~34-35 oC) overnight
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