Team:BYU Provo/Notebook/Cholera - Enzymes/Protocols
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5. Add 500 ul LB to the reaction and incubate at 37°C for 30 minutes.<br> | 5. Add 500 ul LB to the reaction and incubate at 37°C for 30 minutes.<br> | ||
6. Plate 100 ul of cell on appropriate antibiotic plate and incubate at 37°C overnight. | 6. Plate 100 ul of cell on appropriate antibiotic plate and incubate at 37°C overnight. | ||
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+ | <font size="4">'''Verifying Clones with Colony PCR'''</font> | ||
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+ | 1. Add 1 colony to 50 uL of water in a PCR tube. Do this by using a pipet tip and by carefully picking an isolated colony.<br> | ||
+ | 2. Briefly vortex the PCR tube and streak the solution to a marked section of an LB plate to allow you to go back to the correct colony once the correct product is identified.<br> | ||
+ | 3. Boil the solution in the PCR tube for 5 minutes in the PCR machine. The DNA is now ready to use as a template.<br> | ||
+ | 4. Repeat steps 1-3 for seven more colonies to give a total of eight colonies.<br> | ||
+ | 5. Set up a standard ''Taq'' PCR reaction and run for at least 25 cycles.<br> | ||
+ | 6. Verify the inserts by running 5 uL of product out on agarose gel. For 2 or 3 clones that test positive for insert, clean up the PCR products and sequence.<br> | ||
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<u>Biofilm Prep</u> | <u>Biofilm Prep</u> | ||
- | #Overnight culture of V. cholerae grown in | + | #Overnight culture of V. cholerae grown in SSW LB at 30 °C for 72 hrs. |
- | #20 ml of culture added to 180 ml of | + | #20 ml of culture added to 180 ml of SSW LB for dilution |
- | #Fill each well of a 96-well plate with 200 ul of | + | #Fill each well of a 96-well plate with 200 ul of SSW LB |
#To inoculate wells, immerse replicator pins in the diluted bacterial suspension for 30 seconds, then lower replicator pins into wells and agitate for 15 seconds. | #To inoculate wells, immerse replicator pins in the diluted bacterial suspension for 30 seconds, then lower replicator pins into wells and agitate for 15 seconds. | ||
#Plates are covered and incubated with shaking at 30 °C for 24 hrs. | #Plates are covered and incubated with shaking at 30 °C for 24 hrs. | ||
- | #Every 10-12 hrs during the incubation period, spent nutrients are pipetted from wells and replaced with fresh | + | #Every 10-12 hrs during the incubation period, spent nutrients are pipetted from wells and replaced with fresh SSW LB |
<u>Enzyme Treatment</u> | <u>Enzyme Treatment</u> | ||
#At 24 hrs, planktonic suspensions and nutrient solutions are aspirated and wells are rinsed by carefully removing and adding liquid from the side of the well using a micropipette. Wells are washed four times. | #At 24 hrs, planktonic suspensions and nutrient solutions are aspirated and wells are rinsed by carefully removing and adding liquid from the side of the well using a micropipette. Wells are washed four times. |
Latest revision as of 19:48, 14 October 2013
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Cholera-Enzyme Protocols
Synthetic Seawater (SSW) LB recipe
1. To 950 ml distilled H2O add
2. Adjust pH to 7.8
4. Fill to 1000 ml and autoclave
V. cholerae Biofilm growth
1. Start a 5 ml overnight of V. cholerae in SSW LB and incubate at 30° for 72 hours. Phusion PCR (50ul reaction)
1. For each sample add:
2. Set the PCR machine to run the following cycle with the middle three phases being repeated 35 times
Hold at 4°C 3. Store in -20°C freezer
Taq polymerase PCR (50ul reaction)
1. For each sample add:
2. Set the PCR machine to run the following cycle with the middle three phases being repeated 35 times
Hold at 4°C
Restriction Digest (50ul reaction)
1. For each sample add:
2. Place in 37°C incubator or water bath for 1.5 hours
Ligation
1. For each ligation reaction add
3. Incubate the reaction at room temperature for 30 minutes
Transformations
1. Set two heat blocks at 42°C and 37°C respectively.
Verifying Clones with Colony PCR 1. Add 1 colony to 50 uL of water in a PCR tube. Do this by using a pipet tip and by carefully picking an isolated colony.
Protein Purification
Reagents
1. Grow up 500 mL of yeast expressing His-tagged PSK (Grow in PSK activating conditions Gal, Raff, etc.).
Biofilm Assay Protocol
Biofilm Prep
Enzyme Treatment
Absorbance Readings
Final V. cholerae Biofilm Degradation Assay
1. In a culture tube, add 50 ul overnight seed to 1 ml SSW LB in culture tube. (Set up three cultures for each time interval and concentration of enzyme to be assayed.) We chose to test 2.5 ul, 12.5 ul, and 25 ul at time = 0, time = 0 & 42, time = 42, and time = 48.
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