Difference between revisions of "Team:WPI Worcester/Experiments"

(Prototype team page)
 
Line 2: Line 2:
 
<html>
 
<html>
  
−
<div class="column full_size">
+
<div>
  
−
<h1>Experiments</h1>
+
<h2> LB Media </h2>
−
<p>Describe the research, experiments, and protocols you used in your iGEM project. These should be detailed enough for another team to repeat your experiments.</p>
+
  
−
<p>
+
<h3> Material </h3>
−
Please remember to put all characterization and measurement data for your parts on the corresponding Registry part pages.
+
<ul>
−
</p>
+
<li> 2 Liter Flask</li>
 +
<li> LB Broth Powder</li>
 +
<li> DI Water </li>
 +
<li> Bottles</li>
 +
<li> Stir Bar </li>
 +
<li> 1 Liter Graduated Cylinder</li>
 +
<li> Balance </li>
 +
<li> Cup </li>
 +
</ul>
  
 +
<h3> Protocol </h3>
 +
<ol>
 +
<li> Place a large stir bar in a 2 liter flask </li>
 +
<li> Place the 2 liter flask on a stir plate </li>
 +
<li> Use a 1 liter graduated cylinder to measure 1 liter of DI water and pour it into the 2 liter flask </li>
 +
<li> Turn on the stir plate </li>
 +
<li> Place a cup on a balance and tare the balance </li>
 +
<li> Use a scoop to measure out 40 grams of LB Broth powder </li>
 +
<li> Carefully add the 40g of LB broth powder to the 2 liter flask </li>
 +
<li> Allow to mix until all of the powder is dissolved </li>
 +
<li> Pour The LB into smaller (250mL) bottles </li>
 +
<li> Leave caps slightly loose and tape with autoclave tape </li>
 +
<li> Autoclave the LB </li>
 +
<li>Tighten caps </li>
 +
</ol>
 
</div>
 
</div>
  
  
 +
<div>
 +
<h2> LB Agar </h2>
  
−
<div class="column two_thirds_size">
+
<h3> Materials </h3>
−
<h3>What should this page contain?</h3>
+
 
<ul>
 
<ul>
−
<li> Protocols </li>
+
<li> 2 Liter Flask </li>
−
<li> Experiments </li>
+
<li> Stir plate </li>
−
<li> Documentation of the development of your project </li>
+
<li> Petri Dishes </li>
 +
<li> 50℃ Water Bath </li>
 +
<li> Bunsen Burner </li>
 +
<li> Paper Towel </li>
 +
<li> Colored Sharpie </li>
 +
<li> Tinfoil </li>
 +
<li> DI Water </li>
 +
<li> Balance </li>
 +
<li> Cup </li>
 +
<li> Stir bar </li>
 
</ul>
 
</ul>
  
 +
<h3> Protocol </h3>
 +
<ol>
 +
<li> Tare the balance with a cup on it </li>
 +
<li> Measure 40 grams of LB Agar into the cup </li>
 +
<li> Put 1 liter of water in a 2 liter flask on a stir plate </li>
 +
<li> Add the LB agar into the flask and a stir bar into the flask </li>
 +
<li> Allow to mix until powder is dissolved </li>
 +
<li> Put tinfoil on the top of the flask and add a strip of autoclave tape </li>
 +
<li> Autoclave the flask </li>
 +
<li> Mix the flask again </li>
 +
<li> Place the flask in 50℃ water bath for 30 minutes to 2 hours </li>
 +
<li> Add antibiotic to the flask (1mL to 1L) </li>
 +
<li> Prep empty petri dishes in stack of 5 for easy pouring </li>
 +
<li> Label petri dishes with appropriate color that corresponds to antibiotic  </li>
 +
<li> When ready to start pouring, light the bunsen burner and retrieve the flask from the water bath </li>
 +
<li> Wrap a paper towel around the neck of the flask to avoid it getting slippery while pouring </li>
 +
<li> Before pouring each stack of 5 petri dishes, place the lip of the flask into the flame of the bunsen burner to ensure it is sterile </li>
 +
<li> Pour all of the plates </li>
 +
<li> Let the plates cool overnight </li>
 +
<li> Bag the plates and store in the fridge for up to a few months </li>
 +
</ol>
 
</div>
 
</div>
 +
<div>
 +
<h2> DNA Miniprep </h3>
 +
<h3> Materials </h3>
 +
<ul>
 +
<li> A1 buffer </li>
 +
<li> A2 buffer </li>
 +
<li> A3 buffer </li>
 +
<li> A4 buffer </li>
 +
<li> Elution buffer </li>
 +
<li> Overnight culture of E. Coli </li>
 +
<li> Column </li>
 +
<li> Microfuge tube </li>
  
−
<div class="column third_size">
+
</ul>
−
<div class="highlight decoration_A_full">
+
 
−
<h3>Inspiration</h3>
+
<h3> Protocol </h3>
 +
<ol>
 +
<li> Prepare an overnight culture of the E. coli with desired plasmid (5mL LB + 1 colony + 5uL of desired antibiotic) </li>
 +
<li> Incubate in shaker at 37℃ overnight </li>
 +
<li> Spin down culture in big centrifuge at 3500 rpm for 10 minutes </li>
 +
<li> Dump out supernatant into waste beaker </li>
 +
<li> Resuspend pellet in 250 uL A1 buffer </li>
 +
<li> Move culture into 1.5mL microfuge tube </li>
 +
<li> Add 250 uL A2 buffer </li>
 +
<li> Incubate for 5 minutes (mix well, should be blue and sticky) </li>
 +
<li> Add 300uL of A3 buffer (mix until white and fluffy) </li>
 +
<li> Spin in small centrifuge at max speed for 10 minutes </li>
 +
<li> Set up and label columns while sample is spinning </li>
 +
<li> Move supernatant into the column </li>
 +
<li> Spin in small centrifuge 30 seconds max speed </li>
 +
<li> Dump liquid </li>
 +
<li> Add 600uL of A4 </li>
 +
<li> Spin for 30 seconds at max speed </li>
 +
<li> Dump liquid </li>
 +
<li> Spin for 2 minutes  </li>
 +
<li> Move column into microfuge tube </li>
 +
<li> Add 50uL elution buffer </li>
 +
<li> Spin 1 minute at max speed </li>
 +
<li> Nanodrop to check yield </li>
 +
<li> Plug in the nanodrop </li>
 +
<li> Wash the metal plates with DI water </li>
 +
<li> Dry with kimwipe </li>
 +
<li> Put 2uL elution buffer onto bottom plate  </li>
 +
<li> Run blank </li>
 +
<li> Confirm blank by running elution buffer again </li>
 +
<li> Wipe off plates and then run samples </li>
 +
<li> 2uL of sample and record the ng/uL </li>
 +
</ol>
 +
</div>
 +
<div>
 +
<h2> Transformations </h2>
 +
<h3> Materials </h3>
 
<ul>
 
<ul>
−
<li><a href="https://2014.igem.org/Team:Colombia/Protocols">2014 Colombia </a></li>
+
<li> Competent cells </li>
−
<li><a href="https://2014.igem.org/Team:Imperial/Protocols">2014 Imperial </a></li>
+
<li> 1.5mL microfuge tubes </li>
−
<li><a href="https://2014.igem.org/Team:Caltech/Project/Experiments">2014 Caltech </a></li>
+
<li> Plasmid DNA </li>
 +
<li> Ice bucket </li>
 +
<li> Water Bath (42℃) </li>
 +
<li> SOC Medium </li>
 +
<li> 37℃ shaker </li>
 +
<li> Agar plates </li>
 +
<li> Incubator </li>
 
</ul>
 
</ul>
−
</div>
+
 
 +
<h3> Protocol </h3>
 +
<ol>
 +
<li> Thaw competent cells on ice (~30 minutes) </li>
 +
<li> Pre-chill 1.5mL microfuge tubes </li>
 +
<li> Add 50 uL of competent cells into each microfuge tube </li>
 +
<li> Add 1uL of plasmid DNA into the tubes </li>
 +
<li> Incubate on ice for 30 minutes </li>
 +
<li> Heat shock in a water bath (42℃) for 45 seconds </li>
 +
<li> Incubate on ice for 5 minutes </li>
 +
<li> Add 250uL of SOC medium to each tube </li>
 +
<li> Incubate in shaker at 37℃ for 1 hour </li>
 +
<li> Plate the 250uL of cells onto agar plates </li>
 +
<li> Incubate overnight  </li>
 +
</ol>
 +
</div>
 +
<div>
 +
<h2> NCTC Electroporation </h2>
 +
<ol>
 +
<li> Autoclave 500mL of LB in a 1 liter flask  </li>
 +
<li> Autoclave ~2000mL of DI water (then put in 4℃ fridge) </li>
 +
<li> Make 50mL of overnight culture  </li>
 +
<li> Let incubate overnight </li>
 +
<li> Add the 50mL overnight culture to 500mL LB in flask </li>
 +
<li> Put in 37℃ shaker until the OD is at ~0.4 </li>
 +
<li> Centrifuge in big centrifuge at 3500rpm for 10 minutes at 4℃ </li>
 +
<li> Discard supernatant (dump into waste and tap on paper towel) </li>
 +
<li> Resuspend in 500mL of ice cold ddH2O and spin and dump </li>
 +
<li> Do this rinse three times at least </li>
 +
<li> Resuspend in 1mL Ice cold 10% glycerol  </li>
 +
<li> Make 100uL aliquots (Keep in -80℃ freezer or on ice if electroporation is happening directly after) </li>
 +
<li> Chill 1.5mL microfuge tubes </li>
 +
<li> Add 50uL of cells to the microfuge tube </li>
 +
<li> Add 1uL of plasmid DNA to the microfuge tube </li>
 +
<li> Incubate on ice for 10-30 minutes </li>
 +
<li> Carefully put into the electroporation cuvette  </li>
 +
<li> Get volume to 100uL using ddH2O  </li>
 +
<li> Put cuvette in electroporation apparatus </li>
 +
<li> 2-5 kilovolts  </li>
 +
<li> Add 950uL SOC medium to the electroporation cuvette to rinse the cells out of cuvette </li>
 +
<li> Move cells into 15 mL tube and incubate for 1 hr at 37℃ </li>
 +
<li> Plate the cells on agar plates with appropriate antibiotic  </li>
 +
</ol>
 
</div>
 
</div>
  
 +
<div>
 +
<h2> M9 </h2>
 +
<h3> M9 Salts </h3>
 +
<ol>
 +
<li> 800mL H2O </li>
 +
<li> 64g Na2HPO4-7H2O </li>
 +
<li> 15g KH2PO4 </li>
 +
<li> 2.5g NaCl </li>
 +
<li> 5g NH4Cl </li>
 +
<li> Stir until dissolved  </li>
 +
<li> Adjust to 1000mL with distilled H2O </li>
 +
<li> Separate into 5 200mL aliquots  </li>
 +
<li> Autoclave to sterilize </li>
 +
</ol>
  
−
<div class="clear"></div>
+
<h3> M9 Media </h3>
−
 
+
<ol>
−
 
+
<li> 700mL ddH2O </li>
 +
<li> 200mL M9 Salts (above) </li>
 +
<li> 2mL MgSo4 (sterile) </li>
 +
<li> 20mL of 20% glucose (or 20% glycerol)  </li>
 +
<li> 100uL of 1M CaCl2  </li>
 +
<li> 1mL of sterile, filtered 1000x L-Arginine </li>
 +
<li> Adjust to 1000mL using ddH2O  </li>
 +
</ol>
 +
</div>
  
  
 
</html>
 
</html>

Revision as of 15:18, 12 October 2018

LB Media

Material

  • 2 Liter Flask
  • LB Broth Powder
  • DI Water
  • Bottles
  • Stir Bar
  • 1 Liter Graduated Cylinder
  • Balance
  • Cup

Protocol

  1. Place a large stir bar in a 2 liter flask
  2. Place the 2 liter flask on a stir plate
  3. Use a 1 liter graduated cylinder to measure 1 liter of DI water and pour it into the 2 liter flask
  4. Turn on the stir plate
  5. Place a cup on a balance and tare the balance
  6. Use a scoop to measure out 40 grams of LB Broth powder
  7. Carefully add the 40g of LB broth powder to the 2 liter flask
  8. Allow to mix until all of the powder is dissolved
  9. Pour The LB into smaller (250mL) bottles
  10. Leave caps slightly loose and tape with autoclave tape
  11. Autoclave the LB
  12. Tighten caps

LB Agar

Materials

  • 2 Liter Flask
  • Stir plate
  • Petri Dishes
  • 50℃ Water Bath
  • Bunsen Burner
  • Paper Towel
  • Colored Sharpie
  • Tinfoil
  • DI Water
  • Balance
  • Cup
  • Stir bar

Protocol

  1. Tare the balance with a cup on it
  2. Measure 40 grams of LB Agar into the cup
  3. Put 1 liter of water in a 2 liter flask on a stir plate
  4. Add the LB agar into the flask and a stir bar into the flask
  5. Allow to mix until powder is dissolved
  6. Put tinfoil on the top of the flask and add a strip of autoclave tape
  7. Autoclave the flask
  8. Mix the flask again
  9. Place the flask in 50℃ water bath for 30 minutes to 2 hours
  10. Add antibiotic to the flask (1mL to 1L)
  11. Prep empty petri dishes in stack of 5 for easy pouring
  12. Label petri dishes with appropriate color that corresponds to antibiotic
  13. When ready to start pouring, light the bunsen burner and retrieve the flask from the water bath
  14. Wrap a paper towel around the neck of the flask to avoid it getting slippery while pouring
  15. Before pouring each stack of 5 petri dishes, place the lip of the flask into the flame of the bunsen burner to ensure it is sterile
  16. Pour all of the plates
  17. Let the plates cool overnight
  18. Bag the plates and store in the fridge for up to a few months

DNA Miniprep

Materials

  • A1 buffer
  • A2 buffer
  • A3 buffer
  • A4 buffer
  • Elution buffer
  • Overnight culture of E. Coli
  • Column
  • Microfuge tube

Protocol

  1. Prepare an overnight culture of the E. coli with desired plasmid (5mL LB + 1 colony + 5uL of desired antibiotic)
  2. Incubate in shaker at 37℃ overnight
  3. Spin down culture in big centrifuge at 3500 rpm for 10 minutes
  4. Dump out supernatant into waste beaker
  5. Resuspend pellet in 250 uL A1 buffer
  6. Move culture into 1.5mL microfuge tube
  7. Add 250 uL A2 buffer
  8. Incubate for 5 minutes (mix well, should be blue and sticky)
  9. Add 300uL of A3 buffer (mix until white and fluffy)
  10. Spin in small centrifuge at max speed for 10 minutes
  11. Set up and label columns while sample is spinning
  12. Move supernatant into the column
  13. Spin in small centrifuge 30 seconds max speed
  14. Dump liquid
  15. Add 600uL of A4
  16. Spin for 30 seconds at max speed
  17. Dump liquid
  18. Spin for 2 minutes
  19. Move column into microfuge tube
  20. Add 50uL elution buffer
  21. Spin 1 minute at max speed
  22. Nanodrop to check yield
  23. Plug in the nanodrop
  24. Wash the metal plates with DI water
  25. Dry with kimwipe
  26. Put 2uL elution buffer onto bottom plate
  27. Run blank
  28. Confirm blank by running elution buffer again
  29. Wipe off plates and then run samples
  30. 2uL of sample and record the ng/uL

Transformations

Materials

  • Competent cells
  • 1.5mL microfuge tubes
  • Plasmid DNA
  • Ice bucket
  • Water Bath (42℃)
  • SOC Medium
  • 37℃ shaker
  • Agar plates
  • Incubator

Protocol

  1. Thaw competent cells on ice (~30 minutes)
  2. Pre-chill 1.5mL microfuge tubes
  3. Add 50 uL of competent cells into each microfuge tube
  4. Add 1uL of plasmid DNA into the tubes
  5. Incubate on ice for 30 minutes
  6. Heat shock in a water bath (42℃) for 45 seconds
  7. Incubate on ice for 5 minutes
  8. Add 250uL of SOC medium to each tube
  9. Incubate in shaker at 37℃ for 1 hour
  10. Plate the 250uL of cells onto agar plates
  11. Incubate overnight

NCTC Electroporation

  1. Autoclave 500mL of LB in a 1 liter flask
  2. Autoclave ~2000mL of DI water (then put in 4℃ fridge)
  3. Make 50mL of overnight culture
  4. Let incubate overnight
  5. Add the 50mL overnight culture to 500mL LB in flask
  6. Put in 37℃ shaker until the OD is at ~0.4
  7. Centrifuge in big centrifuge at 3500rpm for 10 minutes at 4℃
  8. Discard supernatant (dump into waste and tap on paper towel)
  9. Resuspend in 500mL of ice cold ddH2O and spin and dump
  10. Do this rinse three times at least
  11. Resuspend in 1mL Ice cold 10% glycerol
  12. Make 100uL aliquots (Keep in -80℃ freezer or on ice if electroporation is happening directly after)
  13. Chill 1.5mL microfuge tubes
  14. Add 50uL of cells to the microfuge tube
  15. Add 1uL of plasmid DNA to the microfuge tube
  16. Incubate on ice for 10-30 minutes
  17. Carefully put into the electroporation cuvette
  18. Get volume to 100uL using ddH2O
  19. Put cuvette in electroporation apparatus
  20. 2-5 kilovolts
  21. Add 950uL SOC medium to the electroporation cuvette to rinse the cells out of cuvette
  22. Move cells into 15 mL tube and incubate for 1 hr at 37℃
  23. Plate the cells on agar plates with appropriate antibiotic

M9

M9 Salts

  1. 800mL H2O
  2. 64g Na2HPO4-7H2O
  3. 15g KH2PO4
  4. 2.5g NaCl
  5. 5g NH4Cl
  6. Stir until dissolved
  7. Adjust to 1000mL with distilled H2O
  8. Separate into 5 200mL aliquots
  9. Autoclave to sterilize

M9 Media

  1. 700mL ddH2O
  2. 200mL M9 Salts (above)
  3. 2mL MgSo4 (sterile)
  4. 20mL of 20% glucose (or 20% glycerol)
  5. 100uL of 1M CaCl2
  6. 1mL of sterile, filtered 1000x L-Arginine
  7. Adjust to 1000mL using ddH2O