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Team:Washington/Protocols
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Protocols
Media, Plates, and Solutions
40% and 20% Glucose
40g for 40% or 20g for 20% of Glucose
Mix in 100mL dH2O
Sterile filter into a 150mL bottle
20% Glycerol
20g Glycerol (Liquid)
Mix in 100mL dH2O
Sterile filter into a 150mL bottle
Competent Cell Media Buffer (CCMB)
100g Glycerol (liquid)
10mL x 1M Potassium Acetate
11.8g CaCl2*H2O
4g MnCl2
2g MgCl2
Mix in 1L dH2O
Sterile filter or autoclave (20min at 121C and 20psi) in a 1L bottle
Luria Broth (LB)
10g tryptone
5g yeast extract
10g NaCl
1000mL dH2O
Autoclave in two 500 ml bottle (20 min at 121C and 20psi)
*If using antibiotics create a separate aliquot
LB-Agar
1000ml LB as above
15g agar
1L dH2O
Autoclave in two 500mL bottles (20 min at 121C at 20psi)
*If using antibiotics create a separate aliquot
Super Optimal Broth (SOB)
20g BactoTryptone
5g BactoYeast Extract
10mL x 1M NaCl
2.5mL x 1M KCl
Mix in 1L of diH2O
Sterile filter or autoclave (20 min at 121C and 20psi) in a 1L bottle
Phosphate Buffered Saline (PBS) Solution
8g NaCl
1.44g Na2HPO4
0.8g KCl
0.24g KH2PO4
Mix in 1L of diH2O and buffer to pH 7.4
Sterile filter or autoclave (20 min at 121C and 20psi) in a 1L bottle
Tryptone Phosphate Buffer (TB)
Mix the following in a 1L bottle:
6g Tryptone
12g Yeast Extract
2mL Glycerol
500mL of diH2O
Autoclave (liquid cycle LOOK UP SPECIFICATIONS)
Cool and add 5mL of 100X Potassium Phosphate Salts (17mM KH2PO4 and 72mM K2HPO4)
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.
Selective Dropout media, C-Uracil and C-Histidine (C-Ura and C-His)
Synthesized by the Yeast Resource Center at the University 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
Add dilute HCl to 7.4pH
*Alternatively add slightly less than 250mL of PBS in order to buffer the solution to the appropriate volume.
Basic Cloning
Polymerase Chain Reactions
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 water to 50μl
Conduct the reaction in a Thermocycler, adjusting anneal temperature and extension time accordingly.
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 Water to 50uL
Incubate the reaction for 1hr
Heat inactive the the reaction at the appropriate temperature
Notes: Add the restriction enzyme(s) to the reaction last
Thaw the restriction enzyme(s) on ice to improve shelf life
Ligation
T4 DNA Ligase and Buffer was purchased from New England BioLabs Corporation.
1. Prepare the following in a 0.2mL mircocentrifuge tube:
50.0ng Vector DNA*
37.5ng Vector DNA*
2uL 10X T4 DNA Ligase Buffer
1uL T4 DNA Ligase
Add diH2O to 20uL
2. Incubate the reaction at room temperature for 10-30 minutes or at 16C overnight.
3. Heat inactivate at 65C for 10 minutes.
4. Chill on ice before starting a transformation reaction.
* The exact amount of DNA is dependant on the number of base pairs. In order to conduct a proper reaction consult the New Englan Biolab Ligation Calculator at:
http://nebiocalculator.neb.com/#!/
Escherichia coli Protocols (XL1-Blue and XL10-Gold)
Chemically 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 flasks 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 gentle 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 cryogenic vials or 1.5mL centrifuge tubes.**
15. Store the competent cell aliquots at -80C.
*Streak in such a way that there should be individual colony growth and no clumps after the incubation.
**We did this in a -20C cold room and using a automated repeater pipette.
**The volume of each aliquot depends on the number of transformations you intend to do at a time.
***After removing the cells from incubation keep them on ice or as cold as possible.
Chemically Competent Cell Transformations
1. Thaw competent E.coli cells on ice (XL1-Blue or XL10-Gold)*
2. Add 50uL of competent cells to sterile 15mL conical centrifuge tubes
3. Add 1uL (~100-200ng)* of the miniprep to each culture tube
4. Equilibrate the cells on ice for 10 min
5. Heat shock the cells at 42C for 30-45 seconds**
6. Immediately place the cells back on ice for 3 min
7. Add 250 uL LB media without antibiotics and shake at 250 rpm and 37C for 30 min
8. Spread 10uL and 290uL on an appropriate LB-antibiotic plate
9. Invert the plate and incubate at 37C overnight
*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.
** Do not heat shock for an extended duration as this may damage and/or kill your cells.
Overnights
1. In a 14mL round-bottom tube, add 3-5mL of LB and an appropriate volume of antibiotic(s).
2. Swipe several individual colonies, do not collect satelites or colony clumps, with a pipette tip.
3. Swirl the colony tip in the tube, there should be no visible cell clumps.
4. Incubate and shake the tube at 37C at 250rpm for 12-16 hours and no longer than 20 hours.
DNA-Extraction and mini-preps
All DNA mini-preps were prepared using EPOCH minikits and following the supplied protocols.
Glycerol Stocks
1. Take 1-2mL from an overnight culture and transfer into a 1.5mL centrifuge tube.
2. Spin down the culture at 3000rpm for 3 minutes.
3. Decant the supernatant.
4. Resuspend the cells in 500uL of 40% Glycerol and 500uL of LB(no antibiotics) or water.
5. Transfer the resuspension to a cryogenic vial.
6. Store the glycerol stock at -80C.
Saccharomyces cerevisiae (PYE1 Yeast)
Chemically Competent Cell Culturing
This process take 4 days in lab with a 1 day wait for incubation.
Day 1:
1. Streak yeast cells onto a YPD plate.*
2. Invert the plate and incubate at 30C for 2 days.
Day 3:
1. Add 50mL of YPD liquid media into a 250mL baffle flask.
2. Swipe as many individual colonies as you can see into the YPD media.**
3. Incubate and shake the culture at 30C at 250rpm overnight approximately 24 hours.
Day 4:
1. Take an optical density measurement.
2. In three 250mL baffle flask add the portions of the overnight liquid culture.
3. Dilute each culture to approximately 0.4 optical density with YPD.
4. Incubate and shake the cultures at 30C at 250rpm until the optical density reaches 1.2-1.6.
5. Collect each culture into separate 50ml flat-bottomed centrifuge tubes.
6. Spin down the cells at 4000g for 5 minutes at 4C.
7. Decant the supernatant.
8. Resuspend the cells in 100mL total for all three culture of dH2O.
9. Combine the suspensions into two 50mL flat-bottomed centrifuge tubes.
10. Spin down the cells as above.
11. Decant the supernatant.
12. Resuspend each in 3mL of 100mM Lithium Acetate.
13. Transfer both cultures into a single 15mL conical centrifuge tube.
14. Spin down the cells at 3000rpm for 5 minutes.
15. Resuspend the cells in 0.75mL of 100mM Lithium Acetate, total volume is roughly 2mL.
16. Qualitatively bring up the volume to 3.5mL by adding 40% Glycerol.
17. Aliquot the cells into 1.5mL centrifuge tubes or 1.7mL cryogenic vials.***
*Streak in such a way that there are individual colonies visible on the plate without clumps or satellite colonies.
**Collect only individual visible colonies. Do not collect clumps or satellite colonies.
***The volume of aliquots depends on the number to transformations you intend to do at a time.
Chemically Competent Transformations
This protocol assumes a 50uL aliquot of yeast competent cells were made.
Furthermore, this protocol prepares enough cells for 6 yeast transformations.
1. Add the following to 50uL of yeast competent cells:
240uL of Polyethylene Glycol - 3350 (PEG-3350)
36uL of 1M Lithium Acetate
32uL of dH2O
2. Mix the mixture by gentle pipetting or vortexing.
3. Aliquot 59uL of the mixture into a 0.2mL microcentrifuge tube.
4. Add 1uL (~100-200ng) of DNA.
5. Mix the mixture by gentle pipetting or vortexing.
6. Incubate the mixture at 30C for 30 minutes.
7. Heat shick the mixture at 42C for 20 minutes.
8. Spin down the cells in a microcentrifuge for ~1 minute.
9. Decant the supernatant.
10. Resuspend the cell pellets in 200uL of dH2O.
11. Spin down the cells in a microcentrifuge for ~1 minute.
12. Resuspend the cell pellets in 200uL of dH2O.
13. Plate 50-150uL of the mixture onto an appropriate Selective Dropout Media plate.
14. Invert and incubate at 30C for 2 days.
*The exact amount of DNA depends on the transformation efficiency of your competent cells.
Overnight Culturing
1. In a 14mL round-bottomed culture tube add 1.8mL selective dropout media and 0.2mL 20% glucose.
2. Swipe 3 invidually visible yeast colonies and add them to the culture tube media.
3. Incubate and shake at 37C at 250rpm for 2 days.
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%.
Culture Passaging
1. In a 14mL round-bottomed culture tube add 1.8mL selective dropout media and 0.2mL 20% glucose.
2. Take 20-50uL from a previous overnight or passage culture and add it to the culture media.
3. Incubate and shake at 37C at 250rpm for 2 days.
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%.
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.
Glycerol Stocks
1. Take 1-2mL from an overnight culture and transfer into a 1.5mL centrifuge tube.
2. Spin down the culture at 3000rpm for 3 minutes.
3. Decant the supernatant.
4. Resuspend the cells in 500uL of 40% Glycerol and 500uL of Selective Dropout media or water.
5. Transfer the resuspension to a cryogenic vial.
6. Store the glycerol stock at -80C.
Flow Cytometry
Dilutions
1. From an overnight culture measure the optical density at 660nm by making 1:10 dilutions.
2. Take enough culture to make a 1mL aliquot with OD 0.4.
3. Spin down the aliquot in a 1.5mL centrifuge tube at 3000rpm for 3 minutes.
4. Decant the supernatant.
5. Resuspend the cell pellet in 800uL of the appropriate selctive dropout media and 200uL of 20% Glucose.
6. Transfer the new culture to a 14mL falcon tube.
7. Incubate and shake at 30C and 250rpm for at least 6 hours (1.2-1.6 optical density).
Preparations for Analysis using C6 Accuri Flow Cytometer
1. From the dilution previously made, measure the optical density, roughly 1.2-1.6.
2. Make an aliquot of 500uL of the dilution culture in a 1.5mL centrifuge tube.
3. Spin down the aliquot at 3000rpm for 3 minutes.
4. Decant the supernatant.
5. Resuspend the cell pellet in 500uL of PBS(F).
6. Spin down the resuspension at 3000rpm for 3 minutes.
7. Decant the supernatant.
8. Resuspend the cell pellet in another 500uL of PBS(F).*
9. Prepare the C6 Flow Cytometer by running a backflush cycle and a dH2O cycle.
10. Load the sample onto the sip.
11. Run the sample without 100,000 cell count.
12. Repeat for all samples and make sure to change data cells otherwise the old data is earased.
13. Once finished, run a cleaning cycle with Accuri approved cleaning solution, then run a dH2O cycle.
*For special cases do not resuspend all samples, instead resuspend immediately before running the sample through the flow cytometer.
Fluorescence Activated Cell Sorting
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.
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
1. Add 25mL TB and 25uL Kan to a 250mL baffled flask
2. Stab a glycerol stock with a p1000 pipette and swirl in the flask of media
3. Put flask in 37C shaker overnight
Protein Expression
1. Add 500uL 1000x Kanamycin to 500mL TB in 2L baffled flask
2. Transfer 10mL overnight culture to TB
3. Shake at 37C (DO WE NEED THE RPM?)
4. Remove flask from shaker when optical density is between 0.5 and 0.8
5. Allow flask to rest at room temp for 30 min
6. Add 125uL 1M IPTG
7. Shake flask at 18C overnight
Protein Extraction and Purification
1. Transfer cell culture to centrifuge flask
2. Centrifuge culture at 4000g for 10 min
3. Discard supernatant
4. Resuspend pellet in 25mL lysis buffer
5. Add 250uL of 100x PMSF, 250uL of 100mg/mL lysozyme, and 250 uL of 10mg/mL DNAse
6. Sonicate sample with 0.25inch probe for 5 min at 70% amplitude with 20 sec on and off pulses
7. Take 50uL total sample
8. Transfer lysate to SS-34 centrifuge tube
9. Centrifuge for 30 min at 18000g
10. Take 50uL soluble sample
Nickel Nitrotriacetic Acid Chromatography (Nickel-NTA Chromatography)
1. Add 5mL 50%(v/v) nickel resin in ethanol to 25mL column and allow to settle to ~2.5mL(CV)
2. Rinse with 10CV H20
3. Equilibrate with 10CV lysis buffer
4. Load sample onto column
5. Wash column with 15CV lysis buffer
6. Perform 2 additional wash steps with 15CV
7. Elute sample in 10CV elution buffer and collect eluate
8. Take 50uL pure sample
