Team:British Columbia/Notebook QS

From 2010.igem.org

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<h3>Biofilm Quantification Protocol</h3>
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<h3>Lessons Learned</h3>
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<p><b>Day 1</b></p>
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<p>1. PIck an isolated colony from a Trypticase Soy Agar (TSA) plate and incubate in 5 mL of Trypticase Soy Broth (TSB) without shaking overnight</p>
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<p>1. The 3-A method usually works. However, some parts seem harder to join to other parts. In the case of P2 (BBa_I764104), it took 4-5 times as long as expected to join it to 2 different GFP constructs. We haven't found a fix to this problem, even after varying several variables: insert:vector ratio, ligation volume, and changing ligase buffer. </p>
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<p><b>Day 2</b></p>
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<p>2. PCR is a very good technique for verifying inserts. It is not so good for verifying small inserts (e.g. RBS) since the band would barely change. Sometimes, even when the PCR does not show correct bands, further restriction digests and sequencing yields the correct insert. </p>
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<p>1. Vortex the overnight culture and dilute 30 uL of culture by 1:100 in a 1% glucose solution diluted with TSB</p>
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<p>3. It is possible to clone genes directly from non-purified bacterial DNA. Simply pick a colony of cells containing the desired gene in the genome. This is of course harder to do than plasmid DNA. We've found that addition of DMSO (5-10%) helps the PCR (by presumably allowing primers and reagents to better reach the desired gene). This is true in the case of agrAC, where PCR was unsuccessful without the addition of DMSO.</p>In
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<p>2. Vortex to mix bacteria sample</p>
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<p>4. Don't try to measure PCR product concentration using spectrophotometry right after a PCR. The concentration before and after reaction is basically the same because dNTP's also absorb similarly to strands of DNA. After purification, do measure DNA concentration.</p>
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<p>3. Innoculate 200 uL of dilute sample into pre-determined wells of a microtiter plate</p>
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<p>5. Don't be afraid of going to the lab and start working. The best way to learn is through practice and troubleshooting. It also helps build up a good work flow.</p>
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<p>4. Use 1% glucose solution diluted with TSB, but without bacterial sample, as control and test samples in triplicate</p>
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<p>6. Use polymerases with error checking ability (e.g. Phusion) when cloning genes. </p>
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<p>5. Place lid onto microtiter plate and place in a non-shaking incubator for 24 hours at 37C</p>
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<p>7. Check antibiotics before using them. </p>
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<p><b>Day 3</b></p>
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<p>8. DNA is incredibly stable. DNA stocks will last a long time when stored probably, even in water.</p>
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<p>1. Take microtiter plate out of the incubator and decant all liquid from the wells into a biohazard container</p>
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<p>9. Don't be afraid of asking for help or asking to use other lab groups' equipment.</p>
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<p>2. Wash each well with 3 x 300 uL of Phosphate Buffered Saline (PBS) at room temperature</p>
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<p>10. DNA purification kits don't always work very well. Do check with a nanodrop or other method. Pay close attention to contaminants absorbing at other wavelengths (large peak), as this can fool the machine into thinking there is DNA. </p>
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<p>3. Heat fix remaining bacteria through exposure for 1 hour to 60C air using a heat block</p>
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<p>11. Always sequence constructs if possible, even if simply joining 2 registry parts together. It is the only way to be sure the right parts have joined and reduces panic when parts do not work as expected.</p>
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<p>4. Leave plate inverted overnight at room temperature</p>  
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<p><b>Day 4</b></p>
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<p>1. Add 150 uL of 0.1% crystal violet dye diluted with deionized water to each well</p>
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<p>2. Allow staining to occur for 15 minutes</p>
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<p>3. Decant crystal violet dye from each well into a biohazard container</p>  
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<p>4. Wash wells with deionized water until crystal violet is no longer present in the water</p>
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<p>5. Air dry plate</p>
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<p>6. Take OD readings at an absorbance of 550 nm on a plate reader</p>  
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<h3>Optimization</h3>
 
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<p>Initial experiments were conducted to determine the conditions that were suitable for optimal biofilm growth. The original protocol called for incubation of bacteria in media with a glucose concentration ranging from 0.25%-2%. Through our experiments which tested the OD readings of various glucose concentrations, we concluded that 1% glucose media provided the best environment for biofilm growth of <i>S. aureus</i> strains RN4220 and 8325-4. As well, we determined using identical samples of bacteria and media solution that the outer edges of the Greiner 96-well flat bottom microtiter plate showed abnormal growth possibly due to increased exposure to oxygen when compared to the center wells. Therefore, we decided to use only the center wells from C3 to E10 for our experiments as to avoid extraneous variables. Day 3 of the original protocol was altered as it called for the aspiration of liquid from the wells with a micropipette, whereas we simply inverted the plate into a biohazard container as it was decided that the original method often resulted in biofilm disturbance.</p>
 
                    
                    
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Revision as of 06:31, 27 October 2010



Lessons Learned

1. The 3-A method usually works. However, some parts seem harder to join to other parts. In the case of P2 (BBa_I764104), it took 4-5 times as long as expected to join it to 2 different GFP constructs. We haven't found a fix to this problem, even after varying several variables: insert:vector ratio, ligation volume, and changing ligase buffer.

2. PCR is a very good technique for verifying inserts. It is not so good for verifying small inserts (e.g. RBS) since the band would barely change. Sometimes, even when the PCR does not show correct bands, further restriction digests and sequencing yields the correct insert.

3. It is possible to clone genes directly from non-purified bacterial DNA. Simply pick a colony of cells containing the desired gene in the genome. This is of course harder to do than plasmid DNA. We've found that addition of DMSO (5-10%) helps the PCR (by presumably allowing primers and reagents to better reach the desired gene). This is true in the case of agrAC, where PCR was unsuccessful without the addition of DMSO.

In

4. Don't try to measure PCR product concentration using spectrophotometry right after a PCR. The concentration before and after reaction is basically the same because dNTP's also absorb similarly to strands of DNA. After purification, do measure DNA concentration.

5. Don't be afraid of going to the lab and start working. The best way to learn is through practice and troubleshooting. It also helps build up a good work flow.

6. Use polymerases with error checking ability (e.g. Phusion) when cloning genes.

7. Check antibiotics before using them.

8. DNA is incredibly stable. DNA stocks will last a long time when stored probably, even in water.

9. Don't be afraid of asking for help or asking to use other lab groups' equipment.

10. DNA purification kits don't always work very well. Do check with a nanodrop or other method. Pay close attention to contaminants absorbing at other wavelengths (large peak), as this can fool the machine into thinking there is DNA.

11. Always sequence constructs if possible, even if simply joining 2 registry parts together. It is the only way to be sure the right parts have joined and reduces panic when parts do not work as expected.





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