Team:Washington/Protocols

From 2014.igem.org

(Difference between revisions)
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<p>
<p>
40g for 40% or 20g for 20% of Glucose <br>
40g for 40% or 20g for 20% of Glucose <br>
-
Mix in 100mL H2O <br>
+
Mix in 100mL diH2O. <br>
-
Sterile Filter into a 150mL bottle <br>
+
Sterile filter into a 150mL bottle. <br>
</p>
</p>
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<p>
<p>
20g Glycerol (Liquid) <br>
20g Glycerol (Liquid) <br>
-
Mix in 100mL H2O <br>
+
Mix in 100mL diH2O. <br>
-
Sterile Filter into a 150mL bottle <br>
+
Sterile filter into a 150mL bottle. <br>
</p>
</p>
Line 52: Line 52:
<p>
<p>
         100g Glycerol (liquid) <br>
         100g Glycerol (liquid) <br>
-
           10mL x 1M Potassium Acetate <br>
+
           10mL x 1M Potassium acetate (KCH3COO) <br>
-
           11.8g CaCl2*H2O <br>
+
           11.8g Calcium chloride (CaCl2*H2O) <br>
-
           4g MnCl2 <br>
+
           4g Manganese chloride (MnCl2) <br>
-
           2g MgCl2 <br>
+
           2g Magnesium chloride (MgCl2) <br>
 +
<br>
     Mix in 1L diH2O <br>
     Mix in 1L diH2O <br>
-
     Sterile Filter or Autoclave (20min at 121C at 20psi) in a 1L bottle
+
     Sterile filter or autoclave (20min at 121C and 20psi) in a 1L bottle.
</p>
</p>
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5g yeast extract <br>
5g yeast extract <br>
10g NaCl <br>  
10g NaCl <br>  
-
1000 ml diH2O <br>
+
1000mL diH2O <br>
-
Autoclave in two 500 ml bottle (20 min at 121C at 20psi)
+
<br>
-
 
+
Autoclave in two 500ml bottle (20min at 121C and 20psi). <br>
-
                               *If using antibiotics create a separate aliquot
+
                               Note: If using antibiotics create a separate aliquot. <br>
</p>
</p>
-
<h3> LB-Agar </h3>
+
<h3> LB Agar </h3>
<p>
<p>
-
1000ml L.B. as above <br>
+
1000ml LB as above <br>
15g agar <br>
15g agar <br>
-
Mix into 1L diH2O <br>
+
1L diH2O <br>
-
Autoclave in two 500 ml bottles (20 min at 121C at 20psi) <br>  
+
<br>
-
                                  
+
Autoclave in two 500ml bottles (20min at 121C and 20psi). <br>  
-
                              *If using antibiotics create a separate aliquot
+
                                 Note: If using antibiotics create a separate aliquot. <br>
</p>
</p>
-
<h3> Super Optimal Broth (S.O.B.) </h3>
+
<h3> Super Optimal Broth (SOB) </h3>
                          
                          
                         <p>
                         <p>
        20g BactoTryptone <br>
        20g BactoTryptone <br>
         5g BactoYeast Extract <br>
         5g BactoYeast Extract <br>
-
         10mL x 1M NaCl <br>
+
         10mL x 1M Sodium chloride (NaCl) <br>
-
           2.5mL x 1M KCl <br>
+
           2.5mL x 1M Potassium chloride (KCl) <br>
-
     Mix in 1L of diH2O <br>
+
     <br>
-
     Sterile filter or Autoclave (20 min at 121C at 20psi) in a 1L bottle
+
Mix in 1L of diH2O. <br>
 +
     Sterile filter or autoclave (20min at 121C and 20psi) in a 1L bottle. <br>
                         </p>
                         </p>
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<p>
<p>
-
8g NaCl <br>
+
8g Sodium chloride (NaCl) <br>
-
1.44g Na_2HPO_4 <br>
+
1.44g Disodium phosphate (Na2HPO4) <br>
-
0.8g KCl <br>
+
0.8g Potassium chloride (KCl) <br>
-
0.24g KH_2PO_4 <br>
+
0.24g Monopotassium phosphate (KH2PO4) <br>
-
Mix in 1L of diH2O and buffer to pH 7.4 <br>
+
<br>
-
Sterile Filter or Autoclave (20 min at 121C at 20psi) in a 1L bottle
+
Mix in 1L of diH2O and buffer to pH 7.4. <br>
 +
Sterile filter or autoclave (20min at 121C and 20psi) in a 1L bottle. <br>
</p>
</p>
Line 111: Line 114:
20g Bacto Peptone <br>
20g Bacto Peptone <br>
10g Yeast Extract <br>
10g Yeast Extract <br>
-
Mix into 950mL of diH2O in a 1L bottle <br>
+
<br>
-
Autoclave (20min at 121C at 20psi) <br>
+
Mix into 950mL of diH2O in a 1L bottle. <br>
-
Add 50mL 40% Glucose <br>
+
Autoclave (20min at 121C and 20psi). <br>
-
Sterile Filter into a 1L bottle <br>
+
Add 50mL 40% Glucose. <br>
-
+
Sterile filter into a 1L bottle. <br>
-
For long-term liquid media storage, do not add 40% glucose instead add the glucose directly into cell cultures as needed. <br>
+
<br>
-
For YPD-plates add 24g Bacto Agar to the Bacto Peptone and Yeast Extract before autoclaving. <br>
+
For long-term liquid media storage, do not add 40% Glucose instead add the glucose directly into cell cultures. <br>
 +
For YPD-plates add 24g Bacto Agar to the Bacto Peptone and Yeast Extract before autoclaving. <br>
</p>
</p>
-
<h3> Selective Dropout media, C-Uracil and C-Histidine (C-Ura and C-His)</h3>
+
<h3> C-Uracil and C-Histidine </h3>
<p>
<p>
-
Synethesized by the Yeast Resource Center at the Univeristy of Washington's  
+
Synthesized by the Yeast Resource Center at the Univeristy of Washington's  
Department of Genome Sciences and Department of Biochemistry.
Department of Genome Sciences and Department of Biochemistry.
</p>
</p>
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<p>
<p>
-
For maximum effectiveness, final concentration should be 8.5M in PBS <br>
+
For maximum effectiveness, final concentration should be 8.5M in PBS. <br>
-
203g Guanidinium Hyrdogen Chlordie <br>
+
203g Guanidinium Hydrogen Chloride <br>
250mL PBS solution <br>
250mL PBS solution <br>
</p>
</p>
Line 137: Line 141:
<h2> Basic Cloning </h2>
<h2> Basic Cloning </h2>
-
<h3> Polymerase Chain Reactions </h3>
+
<h3> Polymerase Chain Reactions (PCRs) </h3>
<p>
<p>
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1-5uL of 10uM Forward primer <br>
1-5uL of 10uM Forward primer <br>
1-5uL of 10uM Reverse primer <br>
1-5uL of 10uM Reverse primer <br>
-
<250ng of DNA template <br>
+
250ng of DNA template <br>
-
Nuclease-Free Water to 50μl <br>
+
Nuclease-free H2O to 50μl <br>
-
In a thermocyler conduct the reaction...
+
In a thermocycler, conduct the reaction. Set extension time according to size of amplified DNA and annealing temperature according to the Tm of primer. <br>
</p>
</p>
-
+
 +
<h3> Error-prone Polymerase Chain Reaction </h3>
 +
 
 +
<p>
 +
 +
Prepare 50uL reaction: <br>
 +
5uL 10X Mutazyme II Rxn Buffer <br>
 +
1uL 40mM dNTP mix (200uM each final) <br>
 +
1uL 20uM forward primer <br>
 +
1uL 20uM reverse primer <br>
 +
1uL Mutazyme II DNA polymerase (2.5U/uL) <br>
 +
0.01ng template <br>
 +
QS 50uL diH2O <br>
 +
<br>
 +
Thermocycler: <br>
 +
95C, 2min <br>
 +
95C, 30sec <br>
 +
XXC*, 30sec <br>
 +
72C, Xmin** <br>
 +
32 cycles <br>
 +
72C, 10min <br>
 +
4C, hold
 +
<br>
 +
*Adjust annealing temperature according to Tm of primer. <br>
 +
**Adjust extension time according to the length of amplified DNA. <br>
 +
<br>
 +
Note: Use 0.01ng (calculate by insert and not by total plasmid). <br>
 +
Calculate amount of template to use. <br>
 +
(bp for amplified region) / (bp in total plasmid) = % amplified region <br>
 +
(conc of total plasmid) x (% amplified region as a decimal) = conc of amplified region <br>
 +
<br>
 +
Note: Will probably need to dilute. Never pipette less than 0.5uL. <br>
 +
(0.01ng) / (conc of amplified region) = vol to add to PCR  <br>
 +
</p>
<h3> Restriction Endonuclease Reaction (Digestion) </h3>
<h3> Restriction Endonuclease Reaction (Digestion) </h3>
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1ug of DNA <br>
1ug of DNA <br>
5uL of the appropriate 10X New Englan Biolab Buffer <br>
5uL of the appropriate 10X New Englan Biolab Buffer <br>
-
Nuclease-Free Water to 50uL <br>
+
Nuclease-free H2O to 50uL <br>
Incubate the reaction for 1hr <br>
Incubate the reaction for 1hr <br>
-
Heat inactive the the reaction at the appropriate temperature <br>
+
Heat inactivate the reaction at the appropriate temperature. <br>
-
 
+
Run on gel and gel extract digested fragment. <br>
 +
<br>
Notes:  Add the restriction enzyme(s) to the reaction last <br>
Notes:  Add the restriction enzyme(s) to the reaction last <br>
Thaw the restriction enzyme(s) on ice to improve shelf life <br>
Thaw the restriction enzyme(s) on ice to improve shelf life <br>
Line 174: Line 212:
<h3> Ligation </h3>
<h3> Ligation </h3>
-
<h2> <i> Escherichia coli Protocols (XL1-Blue and XL10-Gold) </i> </h2>
 
-
<h3> Chemically Competent Cell Culturing </h3>
+
<h2> <i> Escherichia coli </i> Protocols (XL1-Blue and XL10-Gold) </h2>
 +
 
 +
<h3> Competent Cell Culturing </h3>
<p>  
<p>  
-
 
+
Competent cells take two days to culture and aliquot. <br>
-
Competent Cells take two days to culture and aliquot. <br>
+
Day 1: <br>
Day 1: <br>
-
1. Streak an aliquot of Compentent Cells onto two LB-plates without anti-biotics.* <br>
+
1. Streak an aliquot of compentent cells onto two LB-plates without antibiotics. <br>
2. Incubate at 37C overnight. <br>
2. Incubate at 37C overnight. <br>
 +
<br>
Day 2: <br>
Day 2: <br>
-
1. In two 250mL baffle flask add 50mL of S.O.B. media. <br>
+
1. In two 250mL baffle flask add 50mL of SOB media. <br>
2. Scrape as many single colonies into either flask. <br>
2. Scrape as many single colonies into either flask. <br>
3. Incubate and shake at 37C and 250rpm for 2-3 hours. <br>
3. Incubate and shake at 37C and 250rpm for 2-3 hours. <br>
Line 193: Line 232:
7. Spin down the cells at 2500rpm at 4C for 15 minutes. <br>
7. Spin down the cells at 2500rpm at 4C for 15 minutes. <br>
8. Decant the supernatant. <br>
8. Decant the supernatant. <br>
-
9. Resuspend the cells in 16mL of CCMB by pipetting or gentle vortexing. <br>
+
9. Resuspend the cells in 16mL of CCMB by pipetting or gently vortexing. <br>
      10. Incubate the cells on ice for 20 minutes. <br>
      10. Incubate the cells on ice for 20 minutes. <br>
      11. Spin down the cells at 2500rpm at 4C for 10 minutes. <br>  
      11. Spin down the cells at 2500rpm at 4C for 10 minutes. <br>  
      12. Decant the supernatant. <br>
      12. Decant the supernatant. <br>
      13. Resuspend the cells in 4mL of CCMB. <br>
      13. Resuspend the cells in 4mL of CCMB. <br>
-
      14. Quickly aliquot the cells into 1.7mL cryogenic vials or 1.5mL centrifuge tubes.** <br>
+
      14. Quickly aliquot the cells into 1.7mL cryovials or 1.5mL centrifuge tubes.* <br>
      15. Store the competent cell aliquots at -80C. <br>
      15. Store the competent cell aliquots at -80C. <br>
 +
<br>
 +
Note: After removing the cells from incubation keep them on ice or as cold as possible. <br>
 +
<br>
 +
*We did this in a -20C cold room and using a automated repeater pipette. <br>
-
*Streak in such a way that there should be invidual colony growth and no clumps after the incubation. <br>
 
-
**We did this in a -20C cold room and using a automated repeater pipette. <br>
 
-
**The volume of each aliquot depends on the number of transformations you intend to do at a time. <br>
 
-
***After removing the cells from incubation keep them on ice or as cold as possible. <br>
 
-
 
</p>
</p>
-
<h3> Chemically Competent Cell Transformations </h3>
+
<h3> Competent Cell Transformations </h3>
-
        <p>
+
<p>
-
1.  Thaw competent <i> E.coli </i> cells on ice (XL1-Blue or XL10-Gold)* <br>
+
1.  Thaw competent E. coli cells on ice (XL1-Blue for cloning). <br>
-
2.  Add 50 uL of competent cells to sterile 15 mL conical centrifuge tubes <br>
+
2.  Add 50uL of competent cells to sterile 14mL Falcon culture tubes. <br>
-
3.  Add 1uL (~100-200ng)* of the miniprep to each culture tube <br>
+
3.  Add 1uL of the miniprep to each culture tube. <br>
-
4.  Equilibrate the cells on ice for 10 min <br>
+
4.  Equilibrate the cells on ice for 10 minutes. <br>
-
5.  Heat shock the cells at 42C for 30-45 seconds** <br>
+
5.  Heat shock the cells at 42C for 30-45 seconds. <br>
-
6.  Immediately place the cells back on ice for 3 min <br>
+
6.  Immediately place the cells back on ice for 3 minutes. <br>
-
7.  Add 250 uL LB media without antibiotics and shake at 250 rpm and 37C for 30 min <br>
+
7.  Add 250uL of LB media and shake at 250rpm and 37C for 30 minutes. <br>
-
8.  Spread 10ul and 290uL on an appropriate LB-antibiotic plate<br>
+
8.  Plate the entire volume onto appropriate plate. <br>
-
9.  Invert the plate and incubate at 37C overnight <br>
+
9.  Invert and incubate at 37C overnight. <br>
 +
</p>
-
*The exact amount of DNA to add depends on your cell's transformation efficiency. However, it is acceptable to add a larger amount to increase the number of transformed cells. <br>
+
<h3> Plating </h3>
-
** Do not heat shock for an extended duration as this may damage and/or kill your cells.
+
-
</p>
+
-
+
<h3> Overnights </h3>
<h3> Overnights </h3>
-
 
-
                        <p>
 
-
1. In a 14mL round-bottom tube, add 3-5mL of LB and an appropriate volume of antibiotic(s). <br>
 
-
2. Swipe several individual colonies, do not collect satelites or colony clumps, with a pipette tip. <br>
 
-
3. Swirl the colony tip in the tube, there should be no visible cell clumps. <br>
 
-
4. Incubate and shake the tube at 37C at 250rpm for 12-16 hours and no longer than 20 hours. <br>
 
-
 
-
</p>
 
<h3> DNA-Extraction and mini-preps </h3>
<h3> DNA-Extraction and mini-preps </h3>
-
 
-
                        <p>
 
-
All DNA mini-preps were prepared using EPOCH minikits and following the supplied protocols.
 
-
</p>
 
<h3> Glycerol Stocks </h3>
<h3> Glycerol Stocks </h3>
-
                        <p>
+
<h2> <i> Saccharomyces cerevisiae </i> (PyE1 Yeast) </h2>
-
1. Take 1-2mL from an overnight culture and transfer into a 1.5mL centrifuge tube. <br>
+
-
2. Spin down the culture at 3000rpm for 3 minutes. <br>
+
-
3. Decant the supernatant. <br>
+
-
4. Resuspend the cells in 500uL of 40% Glycerol and 500uL of LB(no antibiotics) or water. <br>
+
-
5. Transfer the resuspension to a cryogenic vial. <br>
+
-
6. Store the glycerol stock at -80C. <br>
+
-
</p>
+
-
<h2> <i> Saccharomyces cerevisiae (PYE1 Yeast)</i> </h2>
+
<h3> Transformations </h3>
-
 
+
-
<h3> Chemically Competent Cell Culturing</h3>
+
-
 
+
-
                        <p>
+
-
This process take 4 days in lab with a 1 day wait for incubation. <br>
+
-
 
+
-
Day 1: <br>
+
-
1. Streak yeast cells onto a YPD plate.* <br>
+
-
2. Invert the plate and incubate at 30C for 2 days. <br>
+
-
 
+
-
Day 3: <br>
+
-
1. Add 50mL of YPD liquid media into a 250mL baffle flask. <br>
+
-
2. Swipe as many individual colonies as you can see into the YPD media.** <br>
+
-
3. Incubate and shake the culture at 30C at 250rpm overnight approximately 24 hours. <br>
+
-
 
+
-
Day 4: <br>
+
-
1. Take an optical density measurement. <br>
+
-
2. In three 250mL baffle flask add the portions of the overnight liquid culture. <br>
+
-
3. Dilute each culture to approximately 0.4 optical density with YPD. <br>
+
-
4. Incubate and shake the cultures at 30C at 250rpm until the optical density reaches 1.2-1.6. <br>
+
-
5. Collect each culture into separate 50ml flat-bottomed centrifuge tubes. <br>
+
-
6. Spin down the cells at 4000g for 5 minutes at 4C. <br>
+
-
7. Decant the supernatant. <br>
+
-
8. Resuspend the cells in 100mL total for all three culture of dH2O. <br>
+
-
9. Combine the suspensions into two 50mL flat-bottomed centrifuge tubes. <br>
+
-
10. Spin down the cells as above. <br>
+
-
11. Decant the supernatant. <br>
+
-
12. Resuspend each in 3mL of 100mM Lithium Acetate. <br>
+
-
13. Transfer both cultures into a single 15mL conical centrifuge tube. <br>
+
-
14. Spin down the cells at 3000rpm for 5 minutes. <br>
+
-
15. Resuspend the cells in 0.75mL of 100mM Lithium Acetate, total volume is roughly 2mL. <br>
+
-
16. Qualitatively bring up the volume to 3.5mL by adding 40% Glycerol. <br>
+
-
17. Aliquot the cells into 1.5mL centrifuge tubes or 1.7mL cryogenic vials.*** <br>
+
-
 
+
-
*Streak in such a way that there are individual colonies visible on the plate without clumps or satellite colonies. <br>
+
-
**Collect only individual visible colonies. Do not collect clumps or satellite colonies. <br>
+
-
***The volume of aliquots depends on the number to transformations you intend to do at a time. <br>
+
-
 
+
-
</p>
+
-
             
+
-
                <h3> Chemically Competent Cell Transformation </h3>
+
-
<h3> Overnight Culturing </h3>
+
<h3> OVernight Culturing and Passaging </h3>
-
 
+
-
                    <p>
+
-
1. In a a 14mL round-bottomed culture tube add 1.8mL selective dropout media and 0.2mL 20% glucose. <br>
+
-
2. Swipe 3 invidually visible yeast colonies and add them to the culture tube media. <br>
+
-
3. Incubate and shake at 37C at 250rpm for 2 days. <br>
+
-
                               
+
-
                                Note: You can also do 3mL cultures (2.7mL S.D. media and 0.3mL 20% glucose) or larger cultures just make sure to dilute the glucose from 20% to 2%.
+
-
 
+
-
</p>
+
-
 
+
-
                <h3> Culture Passaging </h3>
+
-
+
-
                        <p>
+
-
1. In a 14mL round-bottomed culture tube add 1.8mL selective dropout media and 0.2mL 20% glucose. <br>
+
-
2. Take 20-50uL from a previous overnight or passage culture and add it to the culture media.<br>
+
-
3. Incubate and shake at 37C at 250rpm for 2 days. <br>
+
-
 
+
-
Note: You can also do 3mL cultures (2.7mL S.D. media and 0.3mL 20% glucose) or larger cultures just make sure to dilute the glucose from 20% to 2%. <br>
+
-
Note: The exact amount of culture that you take from a previous culture is irrelevant as long as at least 1 living cell is passaged.
+
-
 
+
-
</p>
+
<h3> Glycerol Stocks </h3>
<h3> Glycerol Stocks </h3>
-
                       
 
-
                        <p>
 
-
1. Take 1-2mL from an overnight culture and transfer into a 1.5mL centrifuge tube. <br>
 
-
2. Spin down the culture at 3000rpm for 3 minutes. <br>
 
-
3. Decant the supernatant. <br>
 
-
4. Resuspend the cells in 500uL of 40% Glycerol and 500uL of C-X or water. <br>
 
-
5. Transfer the resuspension to a cryogenic vial. <br>
 
-
6. Store the glycerol stock at -80C. <br>
 
-
</p>
 
<h2> Flow Cytometry and Fluorescence Activated Cell Sorting </h2>
<h2> Flow Cytometry and Fluorescence Activated Cell Sorting </h2>
<h3> Dilutions </h3>
<h3> Dilutions </h3>
 +
<h3> Final Preparations </h3>
<h3> Final Preparations </h3>
 +
<p> Sample Prep:
 +
 +
Spin down samples and negative control (5000 RPM, 1 min), keeping in mind the library size. Aspirate off supernatant. Resuspend in PBSF. Spin down cells. Aspirate off supernatant. Resuspend in PBSF. <br>
 +
FACS: <br>
 +
1. Open the “iGEM Template” file in the FACS Software and change name to current date and sort cycle<br>
 +
2. Make sure the stream is stable (look for green light in bottom right corner). If not, run the Sort Calibration order. <br>
 +
3. Run Negative Control from the PYE1 cells. <br>
 +
    a. Load cells onto carrier and into the machine. Press play button on screen. <br>
 +
    b. Set gate around lower left quadrant of cells to ensure single cell analysis using Forward Scatter Area and Side Scatter Area as your axes. Make sure oval gate covers around 80% of cell population. <br>
 +
    c. Set second gate on the first gated population by double-clicking on the gated population and using Forward Scatter Height and Forward Scatter Width as your axes. You will notice two distinct populations. Try to focus on the single cell portion of the plot. <br>
 +
NOTE: If you see a large portion of the second gated population existing near the upper right edge of the first gate, you may need to enlarge the first gate to fit more of the population. <br>
 +
    d. Press record. Record 100,000 events and stop run. Move to Next Tube. <br>
 +
4. Run first control (Gene clone) <br>
 +
    a. Follow step 3 to run the first control <br>
 +
5. Run first library sample <br>
 +
    a. Follow steps 3b and 3c to set first two gates correctly. <br>
 +
    b. Set final gate for sort which includes top 1% of GFP producers from second gated population. <br>
 +
    c. Use final gate to set up the sort. <br>
 +
    d. Select sort conditions at the bottom of the screen. <br>
 +
    e. Insert and load collection tube. <br>
 +
    f. Record 100,000 events, and sort 10x the library size <br>
 +
6. Run Bleach and diH2O through FACS to avoid cross-contamination. <br>
 +
</p>
<h2> Protein Expression </h2>
<h2> Protein Expression </h2>
Line 342: Line 313:
<h3> Protein Extraction and Purification </h3>
<h3> Protein Extraction and Purification </h3>
-
<h3> Nickel Nitrotriacetic Acid Column Chromatography (Nickel-NTA Chromatography) </h3>
+
<h3> Nickel Nitrotriacetic Acid Chromatography </h3>
-
<h3> Size Exclusion Chromatography (S.E.C.) </h3>
+
<h3> Size Exclusion Chromatography (SEC) </h3>
<h2> Stability Analysis </h2>
<h2> Stability Analysis </h2>

Revision as of 18:39, 12 October 2014

UW Homepage Official iGEM website

Contents

Protocols

Media, Plates and Solutions

40% and 20% Glucose

40g for 40% or 20g for 20% of Glucose
Mix in 100mL diH2O.
Sterile filter into a 150mL bottle.

20% Glycerol

20g Glycerol (Liquid)
Mix in 100mL diH2O.
Sterile filter into a 150mL bottle.

Competent Cell Media Buffer (CCMB)

100g Glycerol (liquid)
10mL x 1M Potassium acetate (KCH3COO)
11.8g Calcium chloride (CaCl2*H2O)
4g Manganese chloride (MnCl2)
2g Magnesium chloride (MgCl2)

Mix in 1L diH2O
Sterile filter or autoclave (20min at 121C and 20psi) in a 1L bottle.

Luria Broth

10g tryptone
5g yeast extract
10g NaCl
1000mL diH2O

Autoclave in two 500ml bottle (20min at 121C and 20psi).
Note: If using antibiotics create a separate aliquot.

LB Agar

1000ml LB as above
15g agar
1L diH2O

Autoclave in two 500ml bottles (20min at 121C and 20psi).
Note: If using antibiotics create a separate aliquot.

Super Optimal Broth (SOB)

20g BactoTryptone
5g BactoYeast Extract
10mL x 1M Sodium chloride (NaCl)
2.5mL x 1M Potassium chloride (KCl)

Mix in 1L of diH2O.
Sterile filter or autoclave (20min at 121C and 20psi) in a 1L bottle.

Phosphate Buffered Saline (PBS) Solution

8g Sodium chloride (NaCl)
1.44g Disodium phosphate (Na2HPO4)
0.8g Potassium chloride (KCl)
0.24g Monopotassium phosphate (KH2PO4)

Mix in 1L of diH2O and buffer to pH 7.4.
Sterile filter or autoclave (20min at 121C and 20psi) in a 1L bottle.

Yeast Extract Peptone Dextrose (YPD)

20g Bacto Peptone
10g Yeast Extract

Mix into 950mL of diH2O in a 1L bottle.
Autoclave (20min at 121C and 20psi).
Add 50mL 40% Glucose.
Sterile filter into a 1L bottle.

For long-term liquid media storage, do not add 40% Glucose instead add the glucose directly into cell cultures.
For YPD-plates add 24g Bacto Agar to the Bacto Peptone and Yeast Extract before autoclaving.

C-Uracil and C-Histidine

Synthesized by the Yeast Resource Center at the Univeristy of Washington's Department of Genome Sciences and Department of Biochemistry.

Guanidinium Hydrogen Chloride

For maximum effectiveness, final concentration should be 8.5M in PBS.
203g Guanidinium Hydrogen Chloride
250mL PBS solution

Basic Cloning

Polymerase Chain Reactions (PCRs)

All PCRs were done using a standard 50uL reaction volume.
PCRs were done using GoTaq Green Master Mix 2X purchased from PROMEGA Corporation.
Protocols for the PROMEGA GoTaq Green Master Mix 2X:
Mix the following in a 0.2mL PCR tube on ice:
25uL GoTaq® Green Master Mix 2X
1-5uL of 10uM Forward primer
1-5uL of 10uM Reverse primer
250ng of DNA template
Nuclease-free H2O to 50μl
In a thermocycler, conduct the reaction. Set extension time according to size of amplified DNA and annealing temperature according to the Tm of primer.

Error-prone Polymerase Chain Reaction

Prepare 50uL reaction:
5uL 10X Mutazyme II Rxn Buffer
1uL 40mM dNTP mix (200uM each final)
1uL 20uM forward primer
1uL 20uM reverse primer
1uL Mutazyme II DNA polymerase (2.5U/uL)
0.01ng template
QS 50uL diH2O

Thermocycler:
95C, 2min
95C, 30sec
XXC*, 30sec
72C, Xmin**
32 cycles
72C, 10min
4C, hold
*Adjust annealing temperature according to Tm of primer.
**Adjust extension time according to the length of amplified DNA.

Note: Use 0.01ng (calculate by insert and not by total plasmid).
Calculate amount of template to use.
(bp for amplified region) / (bp in total plasmid) = % amplified region
(conc of total plasmid) x (% amplified region as a decimal) = conc of amplified region

Note: Will probably need to dilute. Never pipette less than 0.5uL.
(0.01ng) / (conc of amplified region) = vol to add to PCR

Restriction Endonuclease Reaction (Digestion)

All restriction enzyme reactions were done using a 50ul reaction volume.
Restriction enzymes and buffers were purchased from New England Biolabs Incorporated.
Protocols for various New England Biolab restriction enzyme reactions:
Mix the following in a 0.2mL PCR tube:
1uL of each Restriction Enzyme, add the RE last
1ug of DNA
5uL of the appropriate 10X New Englan Biolab Buffer
Nuclease-free H2O to 50uL
Incubate the reaction for 1hr
Heat inactivate the reaction at the appropriate temperature.
Run on gel and gel extract digested fragment.

Notes: Add the restriction enzyme(s) to the reaction last
Thaw the restriction enzyme(s) on ice to improve shelf life

Ligation


Escherichia coli Protocols (XL1-Blue and XL10-Gold)

Competent Cell Culturing

Competent cells take two days to culture and aliquot.
Day 1:
1. Streak an aliquot of compentent cells onto two LB-plates without antibiotics.
2. Incubate at 37C overnight.

Day 2:
1. In two 250mL baffle flask add 50mL of SOB media.
2. Scrape as many single colonies into either flask.
3. Incubate and shake at 37C and 250rpm for 2-3 hours.
4. Check the optical density of the cells at 550nm after 2 hours.
5. Stop incubation when cultures reach approximately 0.5 optical density.
6. Add the contents of the flask into separate 50mL flat bottomed centrifuge tubes.
7. Spin down the cells at 2500rpm at 4C for 15 minutes.
8. Decant the supernatant.
9. Resuspend the cells in 16mL of CCMB by pipetting or gently vortexing.
10. Incubate the cells on ice for 20 minutes.
11. Spin down the cells at 2500rpm at 4C for 10 minutes.
12. Decant the supernatant.
13. Resuspend the cells in 4mL of CCMB.
14. Quickly aliquot the cells into 1.7mL cryovials or 1.5mL centrifuge tubes.*
15. Store the competent cell aliquots at -80C.

Note: After removing the cells from incubation keep them on ice or as cold as possible.

*We did this in a -20C cold room and using a automated repeater pipette.


Competent Cell Transformations

1. Thaw competent E. coli cells on ice (XL1-Blue for cloning).
2. Add 50uL of competent cells to sterile 14mL Falcon culture tubes.
3. Add 1uL of the miniprep to each culture tube.
4. Equilibrate the cells on ice for 10 minutes.
5. Heat shock the cells at 42C for 30-45 seconds.
6. Immediately place the cells back on ice for 3 minutes.
7. Add 250uL of LB media and shake at 250rpm and 37C for 30 minutes.
8. Plate the entire volume onto appropriate plate.
9. Invert and incubate at 37C overnight.

Plating

Overnights

DNA-Extraction and mini-preps

Glycerol Stocks

Saccharomyces cerevisiae (PyE1 Yeast)

Transformations

OVernight Culturing and Passaging

Glycerol Stocks

Flow Cytometry and Fluorescence Activated Cell Sorting

Dilutions


Final Preparations

Sample Prep: Spin down samples and negative control (5000 RPM, 1 min), keeping in mind the library size. Aspirate off supernatant. Resuspend in PBSF. Spin down cells. Aspirate off supernatant. Resuspend in PBSF.
FACS:
1. Open the “iGEM Template” file in the FACS Software and change name to current date and sort cycle
2. Make sure the stream is stable (look for green light in bottom right corner). If not, run the Sort Calibration order.
3. Run Negative Control from the PYE1 cells.
a. Load cells onto carrier and into the machine. Press play button on screen.
b. Set gate around lower left quadrant of cells to ensure single cell analysis using Forward Scatter Area and Side Scatter Area as your axes. Make sure oval gate covers around 80% of cell population.
c. Set second gate on the first gated population by double-clicking on the gated population and using Forward Scatter Height and Forward Scatter Width as your axes. You will notice two distinct populations. Try to focus on the single cell portion of the plot.
NOTE: If you see a large portion of the second gated population existing near the upper right edge of the first gate, you may need to enlarge the first gate to fit more of the population.
d. Press record. Record 100,000 events and stop run. Move to Next Tube.
4. Run first control (Gene clone)
a. Follow step 3 to run the first control
5. Run first library sample
a. Follow steps 3b and 3c to set first two gates correctly.
b. Set final gate for sort which includes top 1% of GFP producers from second gated population.
c. Use final gate to set up the sort.
d. Select sort conditions at the bottom of the screen.
e. Insert and load collection tube.
f. Record 100,000 events, and sort 10x the library size
6. Run Bleach and diH2O through FACS to avoid cross-contamination.

Protein Expression

Overnight Cultures

Protein Extraction and Purification

Nickel Nitrotriacetic Acid Chromatography

Size Exclusion Chromatography (SEC)

Stability Analysis

Thermal Melts

Guanidinium Hydrogen Chloride Melts

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