Difference between revisions of "Team:Linkoping Sweden/Experiments"

Line 267: Line 267:
 
<li> 72 °C    2 min</li>
 
<li> 72 °C    2 min</li>
 
</ol>
 
</ol>
 +
</div>
 +
<div class="line-separator"></div>
 +
<section class="standard-text-section">
 +
<div class="standard-container">
 +
<h1>
 +
SDS PAGE
 +
</h1>
 +
<h3>
 +
Method
 +
</h3>
 +
 +
<h4>
 +
<ol>
 +
<li>Grow desired product in Falcon tubes</li>
 +
<li>Induce - for every sample there should be a negative control</li>
 +
<li>Incubate in room temperature on a shaking table over night</li>
 +
<li>Check OD (to get a relative protein expression)</li>
 +
<li>Take 500 µl of sample to Eppendorf tube 2 ml</li>
 +
<li>For GroES purification: incubate at 80℃ for 40 min</li>
 +
<li>Centrifuge at 12000 g for 10 min</li>
 +
<li>For samples not incubated at 80℃: Remove supernatant and add 50 µl MilliQ-water</li>
 +
<li>Take 10 µl to a new tube</li>
 +
<li>Add 10 µl SDS sample buffer</li>
 +
<li>For samples incubated at 80℃:  Take 10 µl supernatant to a new tube</li>
 +
<li>Mix with 10 µl SDS sample buffer</li>
 +
<li>Incubate all samples at 95℃ for 5 min. </li>
 +
<li>Take 15 µl of each sample and load on SDS-gel</li>
 +
<li>Run SDS-gel on 150 V until all samples have wandered into the gel and increase the voltage to 200 V</li>
 +
When the loading dye have reached the bottom of the gel, turn it off before it runs out of the gel.</li>
 +
<li>Wash gel with dH2O and set on a shaking table for 5 min. Repeat 3 times.</li>
 +
<li>Color with Comassie G250 for an hour. If the coloring isn’t good enough: use Comassie R250 overnight.</li>
 +
<li>For destaining, a solution of methanol (any grade), 500 ml, dH2O, 400 ml and acetic acid (any grade), 100 ml is needed. Remove the staining solution and replace with the destaining solution, any amount that cover the gel is sufficient. Place on a shaking table for 5-10 mins. Repeat the destaining step until you can read the results.</li>
 +
</ol>
 +
</h4>
 +
 
</div>
 
</div>
 
<div class="line-separator"></div>
 
<div class="line-separator"></div>

Revision as of 05:26, 7 October 2018

LiU iGEM

Experiment protocols

DNA Kit Plate Instructions

To use the DNA in the Distribution Kit, follow these instructions: Note: There is an estimated 2-3ng of DNA in each well, following this protocol, assume that you are transforming with 200-300pg/µL

  1. With a pipette tip, punch a hole through the foil cover into the corresponding well of the part that you want. Make sure you have properly oriented the plate. Do not remove the foil cover, as it could lead to cross contamination between the wells.
  2. Pipette 10µL of dH2O (distilled water) into the well. Pipette up and down a few times and let sit for 5 minutes to make sure the dried DNA is fully resuspended. The resuspension will be red, as the dried DNA has cresol red dye. We recommend that you do not use TE to resuspend the dried DNA.
  3. Transform 1µL of the resuspended DNA into your desired competent cells, plate your transformation with the appropriate antibiotic* and grow overnight.
  4. Pick a single colony and inoculate broth (again, with the correct antibiotic) and grow for 16 hours. Use the resulting culture to miniprep the DNA AND make your own glycerol stock.

Transformation - Double Heat Shock

Method

  1. Resuspend DNA in selected wells in the Distribution Kit with 10 µl dH20. Pipet up and down several times, let sit for a few minutes. Resuspension will be red from cresol red dye.
  2. Label 1.5 ml tubes with part name or well location.
  3. Thaw competent cells on ice: This may take 10-15 min. Dispose of unused competent cells. Do not refreeze unused thawed cells, as it will drastically reduce transformation efficiency.
  4. Pipette 50 µl of competent cells into 1.5 ml tube: 50 µl in a 1.5 ml tube per transformation. Keep all tubes on ice.
  5. Pipette 1 µl of resuspended DNA (10 ng- 1 µg) into 1.5 ml tube: Gently pipette up and down a few times. Keep all tubes on ice.
  6. Pipette 1 µl of control DNA into 2 ml tube: Pipette 1 µl of 10 pg/µl control into your control transformation. Gently pipette up and down a few times. Keep all tubes on ice.
  7. Close 1.5 ml tubes, incubate on ice for 3 h: Tubes may be gently agitated/flicked to mix solution, but return to ice immediately.
  8. Heat shock tubes at 42°C for 45 sec: Timing is critical.
    9. Incubate on ice for 2 min: Return transformation tubes to ice bucket.
  9. Heat shock tubes again at 42°C for 45 sec: Timing is critical.
  10. Pipette 250 µL LB media to each transformation
  11. Incubate at 37°C for 1 hours, shaking at 170 rpm
  12. Pipette 100 µL of each transformation onto agar w/ antibiotic petri plates: Spread with sterilized spreader or glass beads immediately. This helps ensure that you will be able to pick out a single colony.
  13. Spin down cells at 6800 g for 3 mins and discard 800 µL of the supernatant. Resuspend the cells in the remaining 100 µL, and pipette each transformation onto petri plates Spread with sterilized spreader or glass beads immediately. This increases the chance of getting colonies from lower concentration DNA samples.
  14. Incubate transformations overnight (14-18 h) at 37°C: Incubate the plates upside down (agar side up). If incubated for too long, colonies may overgrow and the antibiotics may start to break down; un-transformed cells will begin to grow.
  15. Pick single colonies: Pick single colonies from transformations: do a colony PCR to verify part size, make glycerol stocks, grow up cell cultures and miniprep.
  16. Count colonies for control transformation: Count colonies on the 100 μl control plate and calculate your competent cell efficiency. Competent cells should have an efficiency of 1.5x10^8 to 6x10^8 cfu/µg DNA.

Transformation - Heat Shock

Method

  1. Resuspend DNA in selected wells in the Distribution Kit with 10 µl dH20. Pipet up and down several times, let sit for a few minutes. Resuspension will be red from cresol red dye.
  2. Label 1.5 ml tubes with part name or well location.
  3. Thaw competent cells on ice: This may take 10-15 min. Dispose of unused competent cells. Do not refreeze unused thawed cells, as it will drastically reduce transformation efficiency.
  4. Pipette 50 µl of competent cells into 1.5 ml tube: 50 µl in a 1.5 ml tube per transformation. Keep all tubes on ice.
  5. Pipette 1 µl of resuspended DNA (10 ng- 1 µg) into 1.5 ml tube: Gently pipette up and down a few times. Keep all tubes on ice.
  6. Pipette 1 µl of control DNA into 2 ml tube: Pipette 1 µl of 10 pg/µl control into your control transformation.
  7. Gently pipette up and down a few times. Keep all tubes on ice.
  8. Close 1.5 ml tubes, incubate on ice for 30 min: Tubes may be gently agitated/flicked to mix solution, but return to ice immediately.
  9. Heat shock tubes at 42°C for 45 sec: Timing is critical.
  10. Incubate on ice for 2 min: Return transformation tubes to ice bucket.
  11. Pipette 250 µL LB media to each transformation.
  12. Incubate at 37°C for 1 hour, shaking at 170 rpm.
  13. Pipette 100 µL of each transformation onto agar w/ antibiotic petri plates (see protocol XXX): Spread with sterilized spreader or glass beads immediately. This helps ensure that you will be able to pick out a single colony.
  14. Spin down cells at 6800 g for 3 mins and discard 800 µL of the supernatant. Resuspend the cells in the remaining 100 µL, and pipette each transformation onto petri plates Spread with sterilized spreader or glass beads.
    15. immediately. This increases the chance of getting colonies from lower concentration DNA samples.
  15. Incubate transformations overnight (14-18 h) at 37°C: Incubate the plates upside down (agar side up). If incubated for too long, colonies may overgrow and the antibiotics may start to break down; un-transformed cells will begin to grow.
  16. Pick single colonies: Pick single colonies from transformations: do a colony PCR to verify part size, make glycerol stocks, grow up cell cultures and miniprep.

Agar with chloramphenicol

Method

  1. Pour 15 ml cold liquid agar in a tube. One tube equals one agar plate.
  2. Pipette 15 µl chloramphenicol to the same tube. If more or less agar is used take 1 µl chloramphenicol per ml agar.
  3. Pour the agar solution in to a petri dish. Spread it out evenly. Let dry with lids almost covering the bottom plates.
  4. Apply cells after directions in transformation protocol found here XXX.

Heat shock competent cells, BL21 (DE3)

Method

  1. Take 25 ml LB medium. Put it in a Eppendorf 50 ml tube
  2. Pipette 50 µl cells to the LB medium. In this case BL21 (DE3) from E.coli
  3. Incubate at 37°C, shaking at 170 rpm. For 4-6 hours.
  4. Thaw cells on ice. For 10 mins (keep cold from now on).
  5. Collect the cells by centrifugation. For 5 mins at 1000 rfc
  6. Decant supernatant. Gently resuspend on 10 ml cold 0.1 M CaCl (cells are susceptible to mechanical disruption, so treat them nicely).
  7. Incubate on ice for 20-30 mins
  8. Repeat step 9, but with 2000 rfc.
  9. Discard supernatant. Then gently resuspend with 5 ml cold 0.1 M CaCl2 + 15% Glycerol
  10. Dispense in microtubes (300 μL/tube). Freeze in -80°C.

GenElute™ Plasmid Miniprep Kit

The protocol and material used for plasmid purification was from Sigma-Aldrich

  1. Centrifuge 5 ml of overnight grown competent cells for 1 min at 1200 rcf. Discard the supernatant.
  2. Resuspend cells. Completely resuspend the bacterial pellet with 200 µl of the Resuspension Solution. Vortex or pipette up and down to thoroughly resuspend the cells until homogeneous.
  3. Lyse cells. Lyse the resuspended cells by adding 200 µl of the Lysis Solution. Immediately mix the contents by gentle inversion (6–8 times) until the mixture becomes clear and viscous. Do not vortex. Do not allow the lysis reaction to exceed 5 minutes.
  4. Neutralize. Precipitate the cell debris by adding 350 µl of the Neutralization/Binding Solution. Gently invert the tube 4–6 times. Pellet the cell debris by centrifugation at 12,000 g for 10 min.
  5. Prepare column. Insert a GenElute Miniprep Binding Column into a provided microcentrifuge tube, if not already assembled. Add 500 µl of the Column Preparation Solution to miniprep column and centrifuge at 12,000 g for 1 min.
  6. Discard the flow-through liquid.
  7. Load cleared lysate. Transfer the cleared lysate from step 4 to the column prepared in step 5 and centrifuge at 12,000 g for 1 min. Discard the flow-through liquid.
  8. Wash column. Add 750 µl of the diluted Wash Solution to the column. Centrifuge at 12,000 g for 1 min. Discard the flow-through liquid and centrifuge again at maximum speed for 1 to 2 min without any additional Wash Solution to remove excess ethanol. Discard the flow-through liquid.
  9. Elute DNA. Transfer the column to a fresh collection tube. Add 50 µl molecular biology reagent water to the column. Centrifuge at 12,000 g for 1 min. The DNA is now present in the eluate and is ready for immediate use or storage at –20 °C.

Gel electrophoresis with agarose

Method

  1. Prepare an agarose-gel: Dissolve the agarose in 100 ml 1x TAE buffer. Heat the solution in microwave to make the solution homogeneous (Be careful, the solution must not over boil).
  2. Cool down the solution and pour the agarose into the tank and add 1x TAE buffer until the gel is covered.
  3. Mix DNA samples with purple loading dye from NEB.
  4. Pipette the mixed samples and ladder(NEB) into the wells.
  5. Run at 80 V for 90 minutes.
  6. Carefully remove the gel from the tank and place the gel into a container filled with Ethidium Bromide and shake for 5 minutes.
  7. Place the gel in another container filled with 1x TAE buffer, shake for 1 min.
  8. Use UV light to visualize your DNA fragments.

Q5 Site-Directed Mutagenesis (New England Biolabs)

Primers were designed using www.nebasechanger.neb.com

PCR mix:

  1. 12.5 μL Q5 Hot Start High-Fidelity 2X master mix
  2. 1.25 μL Forward/Reverse primer final conc. 0.5 μM
  3. 1 μL Template DNA (1-25 ng)
  4. 9 μL Nuclease free water

PCR conditions:

  1. 98 °C 30 sec
  2. 98 °C 10 sec (2→ 4 repeated 25 times)
  3. 50-72 °C 10-30 sec
  4. 72 °C 20-30 sec/kb
  5. 72 °C 2 min

KLD reaction:

  1. 1 μL PCR product
  2. 5 μL 2X KLD reaction buffer
  3. 1 μL 10X KLD enzyme mix
  4. 3 μL Nuclease free water
  5. Incubate 5 min

5 μL of the KLD reaction with 50 μL heat shock compatible cells, transformation following whichever chassis you use.

QuikChange II Site-Directed Mutagenesis Kit (Agilent)

Primers were designed using Benchling

PCR mix:

  1. 5 µL 10X reaction buffer
  2. X µL 5–50 ng of dsDNA template
  3. X µL 125 ng of oligonucleotide primer #1
  4. X µL 125 ng of oligonucleotide primer #2
  5. 1 µL dNTP mix
  6. To 50 µL ddH2O

PCR conditions:

  1. 95 °C 30 sec
  2. 95 °C 30 sec (2→ 4 repeated 16 times)
  3. 55 °C 1 min
  4. 68 °C 1 min/kb

Add 1 µl of the Dpn I restriction enzyme (10 U/µl) directly to each amplification reaction.

Gently and thoroughly mix each reaction mixture by pipetting the solution up and down several times. Spin down the reaction mixtures in a microcentrifuge for 1 minute and immediately incubate each reaction at 37°C for 1 hour to digest the parental (i.e., the nonmutated) supercoiled dsDNA.

1 μL of the Dpn I reaction with 50 μL heat shock compatible cells, transformation following whichever chassis you use.

Q5 High-Fidelity 2X Master Mix (New England Biolabs)

Primers were designed using Benchling

PCR mix:

  1. 12.5 μL Q5 High-Fidelity 2X master mix
  2. 1.25 μL Forward/Reverse primer final conc. 0.5 μM
  3. 1 μL Template DNA (1 ng-1 μg)
  4. 9 μL Nuclease free water

PCR conditions:

  1. 98 °C 30 sec
  2. 98 °C 10 sec (2→ 4 repeated 25-35 times)
  3. 50-72 °C 10-30 sec
  4. 72 °C 20-30 sec/kb
  5. 72 °C 2 min

SDS PAGE

Method

  1. Grow desired product in Falcon tubes
  2. Induce - for every sample there should be a negative control
  3. Incubate in room temperature on a shaking table over night
  4. Check OD (to get a relative protein expression)
  5. Take 500 µl of sample to Eppendorf tube 2 ml
  6. For GroES purification: incubate at 80℃ for 40 min
  7. Centrifuge at 12000 g for 10 min
  8. For samples not incubated at 80℃: Remove supernatant and add 50 µl MilliQ-water
  9. Take 10 µl to a new tube
  10. Add 10 µl SDS sample buffer
  11. For samples incubated at 80℃: Take 10 µl supernatant to a new tube
  12. Mix with 10 µl SDS sample buffer
  13. Incubate all samples at 95℃ for 5 min.
  14. Take 15 µl of each sample and load on SDS-gel
  15. Run SDS-gel on 150 V until all samples have wandered into the gel and increase the voltage to 200 V
    16. When the loading dye have reached the bottom of the gel, turn it off before it runs out of the gel.
  16. Wash gel with dH2O and set on a shaking table for 5 min. Repeat 3 times.
  17. Color with Comassie G250 for an hour. If the coloring isn’t good enough: use Comassie R250 overnight.
  18. For destaining, a solution of methanol (any grade), 500 ml, dH2O, 400 ml and acetic acid (any grade), 100 ml is needed. Remove the staining solution and replace with the destaining solution, any amount that cover the gel is sufficient. Place on a shaking table for 5-10 mins. Repeat the destaining step until you can read the results.