Team:Michigan/Oil Sands August September
From 2010.igem.org
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'''Culturing Bacteria in Naphthenic Acids (NA's)''' | '''Culturing Bacteria in Naphthenic Acids (NA's)''' | ||
- | At 12:30 pm the OD600 of the cultures started yesterday were measured and are listed below. These results must be taken with a grain of salt because the cultures were started from the -80C freezer stock with different amounts of | + | At 12:30 pm the OD600 of the cultures started yesterday were measured and are listed below. These results must be taken with a grain of salt because the cultures were started from the -80C freezer stock with different amounts of inoculum: |
*P. putida BH-glu: 0.244 | *P. putida BH-glu: 0.244 | ||
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*E. coli K12 BH-CHCA pH9: 0.0325 | *E. coli K12 BH-CHCA pH9: 0.0325 | ||
- | P. flourescens oilsand seems to love this medium and grew out very | + | P. flourescens oilsand seems to love this medium and grew out very densely! P. putida (oilsand) showed signs of growing. E. coil K12 did not grow at all and pellets of dead cells appeared on the bottom of the culture flask. |
- | From these results we can | + | From these results we can start cultures for the biofilm assay experiment and time the cultures so they all become dense around the same day. If all cultures are started 24 hours in advance they all should saturate by the next day. |
- | The E. coli K12 and P. putida oilsands cultures were placed back in the 30C shaker to keep growing. By 9:30pm the P. putida oilsands cultures had saturated and the E. coli K12 cultures had still not grown | + | The E. coli K12 and P. putida oilsands cultures were placed back in the 30C shaker to keep growing. By 9:30pm the P. putida oilsands cultures had saturated and the E. coli K12 cultures had still not grown. |
As a control for the biofilm assay E. coli K12 will be grown in M9 minimal media with glucose as a carbon source with and without casamino acids. We will not attempt to grow E. coli K12 in Bushnell-Haas media. | As a control for the biofilm assay E. coli K12 will be grown in M9 minimal media with glucose as a carbon source with and without casamino acids. We will not attempt to grow E. coli K12 in Bushnell-Haas media. | ||
- | '''Measuring pH of Bushnell-Haas | + | '''Measuring pH of Bushnell-Haas minimal media with cyclohexane carboxylic acid adjusted to pH 9''' |
- | Today Marc showed us how to use the pH meter in the Lin Lab. Yesterday the pH of the media was estimated by pH paper to be between 8 and 9. The actual pH of the media is 7.35. The pH of the already adjusted media from yesterday was increased by added more 250 uL of 0.1 M NaOH to 5mL of BH CHCA media and the pH ready 7.6 using the pH probe. Another 250 uL of NaOH was added to this | + | Today Marc showed us how to use the pH meter in the Lin Lab. Yesterday the pH of the media was estimated by pH paper to be between 8 and 9. The actual pH of the media is 7.35. The pH of the already adjusted media from yesterday was increased by added more 250 uL of 0.1 M NaOH to 5mL of BH CHCA media and the pH ready 7.6 using the pH probe. Another 250 uL of NaOH was added to this mixture and the pH read 8.8 using the pH probe. pH paper was dotted using this mixture to see the exact color it should turn when the pH is close to 9 (the paper looks more blue than green when the dot first appears). |
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'''Bacterial Growth in pH of 9''' | '''Bacterial Growth in pH of 9''' | ||
- | After determining that 1mL of the supposedly | + | After determining that 1mL of the supposedly already adjusted BH-CHCA media needed 100 uL of 0.1 M NaOH solution per mL to really be close to a pH of 9, new overnight cultures were started of the two pseudomonas oilsand strains in 1 mL of the BH-CHCA pH adjusted media from yesterday with an additional 100 uL of 0.1 M NaOH added. These two cultures were placed in the 30C shaker to grow out overnight at 8:30 pm. |
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'''Miniprep of pBAD take 2''' | '''Miniprep of pBAD take 2''' | ||
- | At 10:45 pm a 5 mL culture of the pBAD biobrick was started from the -80C freezer stock in LB + 25 mg/mL KAN + 1 mM IPTG for a miniprep tomorrow morning | + | At 10:45 pm a 5 mL culture of the pBAD biobrick was started from the -80C freezer stock in LB + 25 mg/mL KAN + 1 mM IPTG for a miniprep tomorrow morning. |
'''PCR of flu operon of E. coli K12''' | '''PCR of flu operon of E. coli K12''' | ||
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The primers ordered on Monday came in today! | The primers ordered on Monday came in today! | ||
- | PCR reagents were picked up from the life science store | + | PCR reagents were picked up from the life science store. |
*dNTP's | *dNTP's | ||
- | **These dNTP's came in four | + | **These dNTP's came in four separate containers individually in a 100 mM solution. The following recipe was used to make the 10 mM dNTP mixture: |
***100 uL of 100 mM dATP | ***100 uL of 100 mM dATP | ||
***100 uL of 100 mM dTTP | ***100 uL of 100 mM dTTP | ||
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***100 uL of 100 mM dCTP | ***100 uL of 100 mM dCTP | ||
***600 uL of ultra pure water | ***600 uL of ultra pure water | ||
- | *Phusion High Fidelity Polymerase | + | *Phusion High Fidelity Polymerase: |
**comes with buffer | **comes with buffer | ||
**used to amplify long pieces of DNA | **used to amplify long pieces of DNA | ||
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'''Gel of flu colony PCR''' | '''Gel of flu colony PCR''' | ||
- | The following gel was run in a 0.7% agarose gel at 85V according to the protocol on the wiki | + | The following gel was run in a 0.7% agarose gel at 85V according to the protocol on the wiki: |
[[Image:8-4-2010_PCR_of_flu_operon.jpg|500px]] | [[Image:8-4-2010_PCR_of_flu_operon.jpg|500px]] | ||
- | For the first 5 cycles the annealing temperature was lowered to the Tm of the 16 or 18 bp that was complementary to the E. coli K12 template. A gradient of temperature was run for the annealing temperatures of the first five | + | For the first 5 cycles the annealing temperature was lowered to the Tm of the 16 or 18 bp that was complementary to the E. coli K12 template. A gradient of temperature was run for the annealing temperatures of the first five cycles as labeled in the gel. |
==8/5/2010== | ==8/5/2010== |
Revision as of 03:37, 26 October 2010