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Team:Washington/Protocols
From 2014.igem.org
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1. Open the “iGEM Template” file in the FACS Software and change name to current date and sort cycle<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> | 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 | + | 3. Run Negative Control from the PyE1 cells. <br> |
a. Load cells onto carrier and into the machine. Press play button on screen. <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> | 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> | ||
Revision as of 18:52, 12 October 2014
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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 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.
