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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 H2O
Sterile Filter into a 150mL bottle
20% Glycerol
20g Glycerol (Liquid)
Mix in 100mL H2O
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 diH2O
Sterile Filter or Autoclave (20min at 121C at 20psi) in a 1L bottle
Luria Broth
10g tryptone
5g yeast extract
10g NaCl
1000 ml diH2O
Autoclave in two 500 ml bottle (20 min at 121C at 20psi)
*If using antibiotics create a separate aliquot
LB-Agar
1000ml L.B. as above
15g agar
Mix into 1L diH2O
Autoclave in two 500 ml bottles (20 min at 121C at 20psi)
*If using antibiotics create a separate aliquot
Super Optimal Broth (S.O.B.)
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 at 20psi) in a 1L bottle
Phosphate Buffered Saline (PBS) Solution
8g NaCl
1.44g Na_2HPO_4
0.8g KCl
0.24g KH_2PO_4
Mix in 1L of diH2O and buffer to pH 7.4
Sterile Filter or Autoclave (20 min at 121C at 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 at 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 as needed.
For YPD-plates add 24g Bacto Agar to the Bacto Peptone and Yeast Extract before autoclaving.
C-Uracil and C-Histidine
Synethesized 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 Hyrdogen Chlordie
250mL PBS solution
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
In a thermocyler conduct the reaction...
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
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 anti-biotics.*
2. Incubate at 37C overnight.
Day 2:
1. In two 250mL baffle flask add 50mL of S.O.B. 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 invidual 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 50 uL of competent cells to sterile 15 mL 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 Cell Transformation
Overnight Culturing and Passaging
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 C-X or water.
5. Transfer the resuspension to a cryogenic vial.
6. Store the glycerol stock at -80C.
