Olivas Lab:Protocols

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High Efficiency E. coli Transformation

Addition of β-Mercaptoethanol (β-ME) to a final concentration of 24 mM has been shown to increase the transformation efficiency of DH5α by 140%. The effect on transformation efficiency may be different when using plasmids other than pUC19. Be sure to use high purity, sterile β-ME at a stock concentration of 1.43 M (most commercial β-ME comes in at 14.3M, so make a 1 in 10 dilution for use with sterile water in a sterile tube). Follow the procedure below:

1. Thaw a 100 µl tube of cells on ice for each transformation you plan to do (plus one or more for a negative control).
2. Add 1.68 µl of 1.43 M β-ME to each tube of cells. Carefully flick the tube 4-5 times to mix cells and β-ME. Do not vortex.
3. Incubate on ice for 10 minutes.
4. Add 1-10 µl containing 1 pg-200 ng of plasmid DNA (or water/negative control solution) to the cell mixture. Carefully flick the tube 4-5 times to mix the cells and DNA. Do not vortex.
5. Place the mixture on ice for 30 minutes. Do not mix.
   • During this time, warm selection plates to 37°C by placing in a 37°C incubator, aliquot 1 mL NZY+ broth per transformation into a sterile tube and set on 42°C heat block.
6. Heat shock each tube of cells at exactly 42°C for exactly 30 seconds. Do not mix.
7. Place on ice for 5 minutes. Do not mix.
8. Pipette 900 µl of 42°C NZY+ broth into the cell mixture.
9. Place at 37°C for 60 minutes. Shake tube vigorously (250 rpm) on its side (using tape to hold it in place) or rotate with end-over-end rocking.
10. Mix the cells thoroughly by flicking the tube and inverting, then perform several (≥3) 10-fold serial dilutions in LB.
    • Alternatively, centrifuge @ 4,000 x RCF for 5 minutes. Remove most of the supernatant and gently resuspend in ~100 µl of remaining supernatant. Plate the entire suspension on the prewarmed selection plate and skip step 11.
11. Spread 50-100 µl of each dilution onto a prewarmed selection plate (usually LB Agar and an antibiotic (e.g., ampicillin/kanamycin/tetracycline)).
12. Incubate overnight (~16h) at 37°C.

NZY+ Recipe

• 10 g of NZ amine (casein hydrolysate)
• 5 g of yeast extract
• 5 g of NaCl
• 1.19g MgCl2
• 3.08g MgSO4•7H20
• Adjust to pH 7.5 using NaOH. Add deionized H2O to a final volume of 1 liter. Autoclave.
• Just before use, add 10 µl of sterile filtered 40% dextrose for every 1 mL of NZY.

Quick E. coli Transformation

For plasmids that are whole, i.e., not a ligation or site-directed experiment, etc., an abbreviated transformation protocol can be used.

This is especially useful for just amplifying a stock plasmid. To date, we have only used this method for plasmids encoding Ampicillin resistance; it may not work with something like Tetracycline resistance which requires a dedicated outgrowth period.

1. Thaw a 100 µl tube of E. coli cells on ice.
2. Add <1 µl of plasmid DNA to the cells.
3. Flick briefly to mix and place back in ice for 5 minutes.
4. Add 900 µl of sterile water to the cells, and pipet up and down briefly to create a homogenous suspension.
5. Plate 100 µl of this dilution on an appropriately selective plate.
6. Incubate overnight (~16h) at 37°C.
7. Pick a single colony from the plate into LB plus appropriate antibiotic.

Preparation of Chemically Competent E. coli Cells (Inoue Method)

The cold growth increases the transformation efficiency. Once the cells have been chilled on ice, perform all steps in a cold room or refrigerated centrifuge! Remember your sterile technique. You will either need a chilled flask shaker or a water bath in a cold room set to 18°C.

1. Streak E. coli on solid media (LB, etc.) for single colonies (i.e., quadrant streak).
2. After single colonies appear (16-20 hrs), pick a single colony from the plate into 25 mL SOC media in a sterile flask. Shake 250-300 rpm at 37°C for 6-8 hrs.
3. Set up three 1L sterile flasks with 250 mL SOC prechilled to 18°C. To one flask, add 10 mL of cells from the previous step, to another add 4 mL, and to the last add 2 mL. Shake at 18°C overnight. (This ensures at least one of the cultures will be at the correct O.D.₆₀₀ the next morning.
4. The next morning, monitor the O.D.₆₀₀ of the cultures. When a culture hits 0.55 O.D.₆₀₀, transfer the entire flask into an ice water bath, and let sit for 10 minutes.
5. Centrifuge the cells at 2,500 x g for 10 minutes in a prechilled sterilized vessel in the cold room or a refrigerated centrifuge.
6. Remove all supernatant.
7. Gently resuspend cells by swirling in 80 mL ice-cold Inoue Resuspension Buffer
8. Centrifuge the cells at 2,500 x g for 10 minutes in a prechilled sterilized 50 mL conicals in the cold room or a refrigerated centrifuge.
9. Remove all supernatant
10. Gently resuspend the cells by swirling in 20 mL ice-cold Inoue Resuspension Buffer (resuspend the cells in one tube with 20 mL, transfer entire contents to the other tube and resuspend with the same solution).
11. Add 1.5 mL of high-quality DMSO and swirl to mix.
12. Aliquot 50 μL into prechilled sterilized 1.7 mL polypropylene tubes.
13. Snap freeze by immersing in liquid nitrogen.
14. Store at -80°C until use.

Recipes

Inoue Resuspension Buffer

• 55 mM MnCl₂
• 15 mM CaCl₂
• 250 mM KCl
• 10mM PIPES (from 0.5M pH 6.7 solution)

Sterilize by passing through a 0.22 μm bottle top filter into an appropriately sized bottle, preferably under a laminar flow hood with good sterile practice. Store at 4°C.

SOC Media

• 0.5% Yeast Extract
• 2% Tryptone
• 10 mM NaCl
• 2.5 mM KCl
• 10 mM MgCl2
• 10 mM MgSO4
• 20 mM Glucose

Sterilize by passing through a 0.22 μm bottle top filter into an appropriately sized bottle, preferably under a laminar flow hood with good sterile practice. Store at room temp.

Hot Phenol RNA Extraction from S. cerevisiae Cell Pellet (The New Hotness Protocol)

Before starting, place aliquot of phenol/ae in 65°C water bath for 10 min and get a bucket of ice. If you have many samples, you may wish to aliquot your phenol/chloroform, chloroform/isoamyl, and 3M NaOAc before starting the extraction process (see step 7).

1. Add 1 mL of 10% SDS to 10 mL of AE buffer in a 15 mL conical.
2. Re-suspend cell pellet in a 2 mL tube in 440 µl of this solution at room temperature.
3. Vortex briefly at room temp (RT).
4. Immediately add 500 µl hot phenol/AE (put in 65°C for 10 min before use, bottom orange layer is the phenol layer) and vortex vigorously for 1 min (vortex in a multitube vortexer if you have several samples).
5. Transfer tubes to floating tube holder and place in 65°C water bath. Incubate at 65°C for 5 min. Briefly vortex every 30 sec.
6. Place tubes on ice for 5 minutes.
7. If you have not already done so, during this time, aliquot your phenol:CHCl3/AE-Na (400 μl), CHCl3:isoamyl alcohol (24:1) (400 μl), and 3M NaOAc (40 μl) into 1.7 mL tubes, one each for each sample/time point. Close and label accordingly.
8. Centrifuge samples from step 5 for 2 min on a standard laboratory microfuge at 21,000 x g at RT.
9. Transfer around 400 μl aqueous supernatant (top layer) to the tube containing phenol:CHCl3/AE-Na (400 μl, two pulls of 200 μl with the P200 micropipette). Make sure not to pull any organic phase (bottom yellow layer) – this will lower your purity. (See step 26)
10. Vortex vigorously for 1 min at RT.
11. Spin down at 21,000 x g for 2 min in a standard laboratory microfuge.
12. Transfer aqueous supernatant (top layer) (around 350 μl, two pulls of 175 μl with the P200 micropipette) to the tube containing CHCl3: isoamyl alcohol (24:1) (400 μl). Make sure not to pull any organic phase (bottom yellow layer) – this will lower your purity. (See step 26)
13. Vortex vigorously for 1 min at RT.
14. Spin down at 21,000 x g for 2 min in a standard laboratory microfuge.
15. Transfer aqueous supernatant (top layer) (around 320 μl, two pulls of 160 μl with the P200 micropipette) to the tube containing 3 M NaOAc (40 μl) and mix briefly. Make sure not to pull any cloudy residue that may be present at the interphase of the two clear layers – this is protein and will lower your purity. (See step 26)
16. Add 1 mL of 100% ethanol (cold). Invert tubes to mix.
17. Place at -80°C for at least 30 min. You can stop here and store RNA in ethanol for more than two weeks.
18. Spin down in the microfuge at 21,000 x g, 10 min at RT. RNA pellet is usually visible.
19. Dump off supernatant, add 200 μl ice-cold 70% EtOH. Vortex briefly and spin down on microfuge 21,000 x g, 5 min at RT.
20. Discard supernatant. Invert tube on Kimwipe and tap gently to remove most of the remainder of ethanol.
21. Place tube in speedvac until no more liquid is observed when flicking the tubes. (~5 minutes)
22. Add 100-200 µl ddH2O (DEPC). Incubate at room temperature for 5 minutes, then vortex briefly to dislodge RNA pellet from the bottom of the tube. Verify visually that the pellet has been dislodged, then place at 55°C for ~10 minutes.
23. Pipet up and down vigorously to resuspend RNA.
24. Quantify immediately and proceed with downstream applications or store in -80°C for more than 2 months.
25. Dispose of all organic phase waste in accordance with institution policy.
26. Remember, it is better to get less volume/yield with higher purity than to try to get every last drop of the aqueous supernatant and risk protein contamination. Do NOT sacrifice yield for purity. Stay away from the bottom phase! 

Recipes

3M NaOAc

• Depc treat and autoclave before use

AE Buffer

• 50 mM NaOAc
• 10 mM EDTA
• pH to 5.0 with acetic acid
• Depc treat and autoclave before use

AE-Na Buffer

• 10 mM NaOAc
• 2 mM EDTA
• 100 mM NaCl
• pH to 6.0 with acetic acid
• Depc treat and autoclave before use

Phenol/AE

• To a volume of liquefied phenol add 0.25% 8-hydroxyquinoline . Equilibrate with an equal volume of AE buffer twice. (To equilibrate, stir vigorously on stir plate for 15 minutes, place in fridge to separate and clear. May take overnight.) Aliquot 35 mL into a polypropylene conical tube and add 10 mL of the AE buffer from the upper layer. Store at -80°C for an indefinite period of time. To use, thaw (65°C or lower) and store at 4°C.

Phenol:CHCl3 /AE-Na

• To a volume of liquefied phenol:CHCl3 (50%:50%) add 0.25% 8-hydroxyquinoline. Equilibrate with an equal volume of AE-Na buffer twice. (To equilibrate, stir vigorously on stir plate for 15 minutes, place in fridge to separate and clear. May take overnight.) Aliquot 35 mL into a polypropylene conical tube and add 10 mL of the AE-Na buffer from the upper layer. Store at -80°C for an indefinite period of time. To use, thaw (65°C or lower) and store at 4°C.

CHCl3: Isoamyl alcohol (24:1)

• Add 1 mL of Isoamyl alcohol to 24 mL of CHCl3. Mix well.

Acknowledgments This protocol for use in the Olivas lab was adapted from www.bio-protocol.org/e209. The following is for citation purposes and comes from the page: This protocol was adapted from and used in Wei and Zheng (2009) and Wei et al. (2009). References 1. Wei, Y. and Zheng, X. F. (2009). Sch9 partially mediates TORC1 signaling to control ribosomal RNA synthesis. Cell Cycle 8(24): 4085-4090. 2. Wei, Y., Tsang, C. K. and Zheng, X. F. (2009). Mechanisms of regulation of RNA polymerase III-dependent transcription by TORC1. EMBO J 28(15): 2220-2230.

How to cite this protocol: Wei, Y. (2012). A Simple Preparation of RNA from Yeast by Hot Phenol for Northern Blot. Bio-protocol 2(12): e209. http://www.bio-protocol.org/e209

Homemade DNA Purifications

Plasmid Miniprep from E. coli

Before starting, thaw a tube of 10 mg/mL RNAseA. Gently flick to mix to make sure the solution is homogenous. All steps are carried out at room temperature.

1. The night before, pick a single E. coli colony containing the plasmid into 2-10 mL of appropriately selective media (usually LB with 100 µg/mL Ampicillin). Plan to extract in 12-16 hours.
2. The next morning, spin down the 2-10 mL culture from night before in a 1.7 mL tube.
3. Discard supernatant.
4. Resuspend the cell pellet in 250 µl Buffer R.
5. Add 2.5 µl of the 10 mg/mL RNAse A. Invert 4-6 times to mix thoroughly.
6. Add 125 µl of Buffer L1.
7. Add 125 µl of Buffer L2.
8. Mix by gentle inversion for 1-2 minutes.
9. Add 300 µl of Buffer N.
10. Mix by gentle inversion for 1 minute.
11. Centrifuge at max speed (≥16,000 x g) for 2-4 minutes.
    • If precipitate is seen floating on top of the supernatant after the centrifugation, repeat the centrifugation (this is usually not an issue).
12. Meanwhile, place a silica column into a 2 mL tube with the lid cut off (the 2 mL tube becomes the collection tube).
13. Transfer the supernatant from step 10 to the spin column, being careful not to transfer any solid precipitate at the bottom of the tube.
14. Allow to sit at room temperature for 1 minute.
15. Centrifuge at max speed (≥16,000 x g) for 1 minute.
    • You may be able to get better yields spinning at low speeds, as low as 100 x g.
16. Remove the spin column and empty the collection tube. Place the spin column back in the collection tube.
17. Add 500 µl of Buffer W1 to the column. Centrifuge at max speed (≥16,000 x g) for 1 minute.
18. Remove the spin column and empty the collection tube. Place the spin column back in the collection tube.
19. Add 750 µl of Buffer W2 to the column. Centrifuge at max speed (≥16,000 x g) for 1 minute.
20. Remove the spin column and empty the collection tube. Place the spin column back in the collection tube.
21. Centrifuge at max speed (≥16,000 x g) for 1 minute to remove any remaining traces of wash.
22. Remove the spin column and discard the collection tube. Place the spin column in a new 1.7 mL tube.
23. Add 50 µl of Buffer E to the column. Let stand 1 minute. Centrifuge at max speed (≥16,000 x g) for 1 minute.
    • You may be able to get better yields by preheating Buffer E to 50°C. Also, doing two elutions with half the volume for each elution may yield more - a lot of DNA will still be left on the column after one elution.
24. Remove the spin column and place in Buffer X.
    • Store the column in Buffer X for at least 24 hours and no more than a month. To reuse the column, rinse the outside thoroughly with distilled water and rinse the inside by centrifuging a full column’s worth of nuclease free water (Depc’d) through three times. After rinsing they can be stored dry.
25. Your plasmid DNA is now in your 1.7 mL tube. Assess the purity and concentration of the plasmid DNA. A purity of <1.75 indicates significant protein contamination, a purity of >1.95 indicates significant RNA contamination. 1.8 is a good number. If the concentrations of your spec’d DNA do not match what you see on a gel, you may have genomic DNA contamination. Try not to let the culture density rise much above 4 or 5 O.D.600.

Plasmid Maxiprep from E. coli Using Miniprep Silica Columns (Miraprep Method)

Before starting, thaw a tube of 10 mg/mL RNAseA. Gently flick to mix to make sure the solution is homogenous. All steps are carried out at room temperature.

1. The night before, pick a single E. coli colony containing the plasmid into 100 mL of appropriately selective media (usually LB with 100 µg/mL Ampicillin). Plan to extract in 12-16 hours.
2. The next morning, spin down the 100 mL culture from night before in two 50 ml conical tubes.
3. Discard supernatant.
4. Resuspend the cell pellet in 5 mL Buffer R.
5. Add 50 µl of the 10 mg/mL RNAse A. Invert 4-6 times to mix thoroughly.
6. Add 2.5 mL of Buffer L1.
7. Add 2.5 mL of Buffer L2.
8. Mix by gentle inversion for 1-2 minutes.
9. Add 6 mL of Buffer N.
10. Mix by gentle inversion for 1 minute.
11. Aliquot 2 ml into 2 ml tubes, enough to handle the entire volume (will be ~18 mL, so 9 tubes plus a balance).
12. Centrifuge at max speed (≥16,000 x g) for 2-4 minutes.
    • If precipitate is seen floating on top of the supernatant after the centrifugation, repeat the centrifugation (this is usually not an issue).
13. Meanwhile, place 10 silica columns into 10 2 mL tubes with the lids cut off (the 2 mL tube becomes the collection tube).
14. Transfer all the supernatant from the tubes in step 12 into one 50 ml conical tube.
15. Add an equal volume of 95% ethanol. Invert to mix.
16. Transfer the solution to the silica columns.
17. Allow to sit at room temperature for 1 minute.
18. Centrifuge at max speed (≥16,000 x g) for 1 minute.
    • You may be able to get better yields spinning at low speeds, as low as 100 x g.
19. Remove the spin column and empty the collection tube. Place the spin column back in the collection tube.
20. Continue adding the solution and repeating the 2 previous steps until all the solution has been passed through the columns. Each column will be processing ~4 mL of solution.
21. Add 500 µl of Buffer W1 to each column. Centrifuge at max speed (≥16,000 x g) for 1 minute.
22. Remove the spin column and empty the collection tube. Place the spin column back in the collection tube.
23. Add 750 µl of Buffer W2 to each column. Centrifuge at max speed (≥16,000 x g) for 1 minute.
24. Remove the spin column and empty the collection tube. Place the spin column back in the collection tube.
25. Centrifuge at max speed (≥16,000 x g) for 1 minute to remove any remaining traces of wash.
26. Remove the spin column and discard the collection tube. Place the spin column in a new 1.7 mL tube.
27. Add 25 µl of Buffer E to the column. Let stand 1 minute. Centrifuge at max speed (≥16,000 x g) for 1 minute.
    • You may be able to get better yields by preheating Buffer E to 50°C. Also, doing two elutions with half the volume for each elution may yield more - a lot of DNA will still be left on the column after one elution.
28. Remove the spin columns and place in Buffer X.
    • Store the column in Buffer X for at least 24 hours and no more than a month. To reuse the column, rinse the outside thoroughly with distilled water and rinse the inside by centrifuging a full column’s worth of nuclease free water (Depc’d) through three times. After rinsing they can be stored dry.
29. Combine all the eluates into one tube. Should have ~250 μL
30. Your plasmid DNA is now in your 1.7 mL tube. Assess the purity and concentration of the plasmid DNA. A purity of <1.75 indicates significant protein contamination, a purity of >1.95 indicates significant RNA contamination. 1.8 is a good number. Expect over a μg of DNA per μL. If the concentrations of your spec’d DNA do not match what you see on a gel, you may have genomic DNA contamination. Try not to let the culture density rise much above 4 or 5 O.D.600.

Plasmid Miniprep from Yeast (Saccharomyces cerevisiae)

1. Grow a 5 mL overnight culture from a single colony in an appropriately selective media.
2. The next day, spin down the entire culture 3,200 x g for 2 minutes.
3. Remove the supernatant from the cells and resuspend in 250 µl Buffer R.
4. Transfer the cell suspension to a 1.7 mL tube.
5. Add 2.5 µl 10mg/ mL RNAse A.
6. Add 100 µl of 0.5mm glass beads to the tube (add glass beads until you hit the 100 µl graduation on the tube).
7. Vortex at max speed for 5 minutes.
8. Proceed from step 6 of the “Plasmid Miniprep from E. coli” protocol, except in step 23 elute in 20 µl. Use 10 µl to transform E. coli. 

Purification of DNA from Agarose Gel Slice

1. On an analytical balance, tare the scale with an empty 1.7 mL tube.
2. Remove the tube, place the excised gel slice in the tube, close, and place back on the scale.
3. Record the mass of the slice in mg.
4. For every mg of agarose gel, add 3 µl of Buffer B (e.g., if your gel slice weighed 120 mg, you would add 360 µl of Buffer B to the tube).
5. Incubate at 50°C for 10 min or until the gel slice has completely dissolved. Vortex the tube gently every minute or so to help dissolve the gel.
   1. Place a silica column in a 2 mL tube with the lid cut off (the 2 mL tube is now the collection tube).
6. Transfer the entire solution onto the silica column.
7. Let stand for 1 minute at room temperature.
8. Centrifuge at max speed (≥16,000 x g) for 1 minute.
   • You may be able to get better yields spinning at low speeds, as low as 100 x g.
9. Remove the spin column and empty the collection tube. Place the spin column back in the collection tube.
10. Add 750 µl of Buffer W3 to the column. Centrifuge at max speed (≥16,000 x g) for 1 minute.
11. Remove the spin column and empty the collection tube. Place the spin column back in the collection tube.
12. Add 500 µl of Buffer W3 to the column. Centrifuge at max speed (≥16,000 x g) for 1 minute.
13. Remove the spin column and empty the collection tube. Place the spin column back in the collection tube.
14. Centrifuge at max speed (≥16,000xg) for 1 minute to remove any remaining traces of wash.
15. Remove the spin column and discard the collection tube. Place the spin column in a new 1.7 mL tube.
16. Add 30 µl of Buffer E to the column. Let stand 1 minute. Centrifuge at max speed (≥16,000 x g) for 1 minute.
    • You may be able to get better yields by preheating Buffer E to 50°C. Also, doing two elutions with half the volume for each elution may yield more - a lot of DNA will still be left on the column after one elution.
17. Remove the spin column and place in Buffer X.
    • Store the column in Buffer X for at least 24 hours and no more than a month. To reuse the column, rinse the outside thoroughly with distilled water and rinse the inside by centrifuging a full column’s worth of nuclease free water (Depc’d) through three times. After rinsing they can be stored dry.
18. Your cleaned DNA is now in your 1.7 mL tube. Assess the purity and concentration of the DNA. A purity of <1.75 indicates significant protein contamination. 1.8 is a good number.

Purification of DNA from PCR or Restriction Digest/Other Enzymatic Reaction

1. Transfer the reaction containing the DNA to a 1.7 mL tube.
2. For every µl of reaction, add 3 µl of Buffer B.
3. Invert several times to thoroughly mix.
4. Place a silica column in a 2 mL tube with the lid cut off (the 2 mL tube is now the collection tube).
5. Transfer the entire solution onto the silica column.
6. Let stand at room temperature for 1 minute.
7. Centrifuge at max speed (≥16,000 x g) for 1 minute.
   • You may be able to get better yields spinning at low speeds, as low as 100 x g.
8. Remove the spin column and empty the collection tube. Place the spin column back in the collection tube.
9. Add 750 µl of Buffer W3 to the column. Centrifuge at max speed (≥16,000 x g) for 1 minute.
10. Remove the spin column and empty the collection tube. Place the spin column back in the collection tube.
11. Centrifuge at max speed (≥16,000 x g) for 1 minute to remove any remaining traces of wash.
12. Remove the spin column and discard the collection tube. Place the spin column in a new 1.7 mL tube.
13. Add 30 µl of Buffer E to the column. Let stand 1 minute. Centrifuge at max speed (≥16,000 x g) for 1 minute.
    • You may be able to get better yields by preheating Buffer E to 50°C. Also, doing two elutions with half the volume for each elution may yield more - a lot of DNA will still be left on the column after one elution.
14. Remove the spin column and place in Buffer X.
    • Store the column in Buffer X for at least 24 hours and no more than a month. To reuse the column, rinse the outside thoroughly with distilled water and rinse the inside by centrifuging a full column’s worth of nuclease free water (Depc’d) through three times. After rinsing they can be stored dry.
15. Your cleaned DNA is now in your 1.7 mL tube. Assess the purity and concentration of the DNA. A purity of <1.75 indicates significant protein contamination. 1.8 is a good number.

Recipes

Buffer R

• 50 mM Tris-Cl pH 8.0
• 10 mM EDTA

Buffer L1

• 2% SDS

Buffer L2

• 0.4M NaOH

Buffer N

• 4.2 M Guanidine-HCl
• 0.9 M potassium acetate
• pH to 4.8 with acetic acid

Buffer W1

• 5M Guanidine-HCL
• 30% 2-Propanol

Buffer W2

• 10mM Tris-Cl, pH 8.0
• 80% Ethanol

Buffer W3

• 10mM Tris-Cl, pH 8.0
• 100mM NaCl
• 1mM EDTA
• 50% Ethanol

Buffer E

• Nuclease Free Water (treating with diethylpyrocarbonate is our method of making nuclease free water)

Buffer X

• 1M HCl

Buffer B

• 6M NaI
• Store at 4°C for no more than 3 months. May develop a yellow color, this does not affect its performance.

Yeast Transformation Protocol (Gietz Lab Best Protocol, with additions from our lab)

Day 1

1. Inoculate the yeast strain into 5 mL of liquid medium (YPD or SC selection medium plus a carbon source) and incubate overnight on a rotary shaker at 200 rpm and 30°C (or 24°C if it is our temperature sensitive background).

Day 2

1. Calculate how much volume of overnight culture is required to make a culture of OD600 0.167 in 50 mL of the same media it was growing in.
2. Make this culture.
3. Incubate the flask on a rotary shaker at 30°C (or 24°C) and 200 rpm.
   • It is important to allow the cells to complete at least two divisions.
   • This will take 3 to 5 hours.
   • This culture will give sufficient cells for 10 transformations. Transformation efficiency (transformants/µg plasmid) remains constant for 3 to 4 cell divisions.
4. When the cells have at least doubled twice, which should take about 4.5 hours (O.D. should be 0.667), aseptically harvest the cells in a 50 mL sterile conical by centrifugation at 3,200 x RCF for 2 min.
   • If the culture is above 0.667, calculate how much less volume it will take to reach the same cell count by using C_iV_i=C_fV_f. For example, if the culture is instead at 0.83, the calculation would be 0.667*50 mL = 0.83*x mL, and x mL = 33.35/0.83 = 41.69 mL.
   • At this time, boil a 1.0 mL sample of 2mg/ mL SSDNA for 5 min and chill in an ice/water bath while harvesting the cells.
5. Discard the supernatant.
6. Wash the cells in 25 mL of sterile water (add 25 mL sterile water and shake to resuspend or vortex vigorously).
7. Centrifuge at 3,200 x RCF for 2 min.
8. Discard supernatnt.
9. Resuspend in 1 mL of sterile water by pipetting up and down and transfer into a sterile 1.7 mL tube.
10. Centrifuge at 3,200 x RCF for 2 min.
11. Discard supernatant.
12. Add water to a final volume of 1.0 mL (using tube volume markings) and pipet up and down to resuspend.
13. Pipette 100 µL samples into sterile 1.5 mL microfuge tubes, one for each transformation and control.
14. Centrifuge at 3,200 x RCF for 2 min.
15. Remove the supernatant.
16. Each tube of cells receives the following reagents/volumes, IN THE ORDER LISTED:
    • 240 µL 50% PEG
    • 50 µL 2mg/ mL SSDNA
    • X µL DNA to be transformed (not to exceed 34 µL total volume)
    • 34-X µL sterile water, where X is the total amount of DNA to be transformed
    • 36 µL 1.0 M LiOAc
17. Pipet up and down to resuspend the cell pellet in the transformation mix.
18. Incubate the tubes in a 42°C water bath for 40 minutes.
    • Note:The optimum time can vary for different yeast strains. Please test this if you need high efficiency from your transformations.
19. Centrifuge at 3,200 x RCF for 2 min.
20. Remove the supernatant and discard.
21. Pipette 1.0 mL of sterile water into each tube; gently resuspend the pellet by pipetting up and down.
    • Alternatively, if transforming in a resistance gene, either recover in media for 3 hours and then plate or plate on media with no antibiotic, let grow overnight, and then replica plate onto media containing antibiotic.
22. Plate 200 µL on at least one plate, and up to 5. Incubate the plates at the required temperature.
    • You may optionally resuspend in 200 µL and plate everything, but this can lead to poor growth due to crowding (we assume).

This protocol was taken almost verbatim from the following website: https://home.cc.umanitoba.ca/~gietz/method.ht mL We have made slight additions to suit our needs, most notably gentler resuspension of the cells throughout the last half (treating the cells too roughly can lead to decreased transformation efficiency).

Simple Yeast Lysis Protocol for SDS-PAGE

This method of lysing yeast cells for total protein is easy (albeit longer than others) and produces nice clean results (in our hands). It is from Zhang et. al., Yeast 2011 28:795-798.

• Reagents Needed:

1. 2X SDS-PAGE Loading Buffer (we like the MilliporeSigma formulation)(You can find a copycat formulation in the recipes section.)
2. 2.0 M LiOAc
3. 0.4 M NaOH
4. Ice

That's it!

• Protocol

1. Harvest 1.5 O.D.₆₀₀ units worth of cells in a 1.7 mL tube by centrifuging at 3,200 x RCF for 2 minutes and then removing and discarding the supernatant.
   • An O.D. units is calculated by multiplying the O.D._nm by the total volume of the culture. For example, a culture that is at O.D.₆₀₀ 0.6 and is 25 mL total would be the equivalent of 15 O.D. units. Therefore, 1.5 O.D. units would be (1.5/0.6) = 2.5 mL. To get into a 1.7 mL tube you may have to do successive spins, removing the supernatant each time.
2. Resuspend the cell pellet in 1 mL 2.0 M LiOAc.
3. Place on ice for 5 minutes.
4. Centrifuge at 3,200 x RCF for 2 minutes and then remove and discard the supernatant.
5. Resuspend the cell pellet in 1 mL 0.4 M NaOH.
6. Centrifuge at 3,200 x RCF for 2 minutes and then remove and discard the supernatant.
7. Resuspend the cell pellet in 100 µL 2X SDS-PAGE Loading Buffer.
8. Boil for 5 minutes at 100°C.
9. Centrifuge at 3,200 x RCF for 2 minutes and then remove and SAVE the supernatant and move to a new tube.
10. Discard the insoluble pellet.
    • You can optionally save the pellet if you suspect your protein may be an insoluble gunky blob and try to resuspend it in something more harsh like urea.
11. Load as much as you want on an SDS-PAGE gel. The original paper called for 10 µL to be loaded, but YMMV.
12. Proceed with Coomassie staining or Western blotting.

Common Reagent Recipes

6X Load Dye for SDS-PAGE Gels

(http://recipes.labguru.com/recipes/laem mLi-buffer-6x-10 mL)

• 1.2g SDS
• 5.92g Glycerol
• 0.6 mL 1M Tris-HCl pH 6.8
• 2.7 mL ddH2O
• 0.93g DTT (Dithiothreitol)
• 6mg Bromophenol Blue

Add all reagents except DTT and Bromophenol Blue into 15 mL conical tube. Warm at 68°C and invert until dissolved (will take a while). Spin down in centrifuge to confirm everything is dissolved. Add DTT, invert to solubilize, then add Bromophenol Blue and invert several times. Spin down in centrifuge to confirm all Bromophenol Blue is dissolved (undissolved Bromophenol Blue will appear orange at the bottom). Final volume should be ~10 mL. Aliquot 250 µl per 1.7 mL microfuge tube, mark “6X PLB”, and store at -20°C. Limit the number of freeze/thaw cycles to preserve integrity of DTT.

2X Laemmli Load Dye for SDS-PAGE Gels (copycat Millipore-Sigma formulation)

• 20ml 10% SDS
• 2mg Bromophenol Blue
• 6.25ml 1M Tris-HCl pH 6.8
• 8.75ml ddH2O
• 10ml glycerol

Add all reagents except glycerol into 50ml conical tube. Invert several times until all bromophenol blue is dissolved, then add glycerol and invert to mix thoroughly. Final volume will be 45ml. 2-mercaptoethanol must be added fresh before use each time; add 100µl 2-mercaptoethanol to each 900µl of this solution to make final 2X solution (mark on the side of the conical that it does NOT have 2-mercaptoethanol in it to remind people to add it. May be stored at room temp.

10X Semi-Dry Western Transfer Buffer (Bjerrum Schafer-Nielsen formulation)(Biorad)

• 58.15 g Tris base
• 29.28 g Glycine
• 800 mL ddH2O

Mix the Tris and Glycine in the 800 mL water until dissolved. pH should be 9.2. Mark “10X Semi-Dry Western Transfer Buffer w/o Methanol”. To make 1X Transfer Buffer, mix 10 mL of 10X buffer, 70 mL ddH2O, and 20 mL methanol, IN THAT ORDER. You *may* optionally add 375 µl of 10% SDS if you have been having trouble blotting proteins. Equilibrate membrane for 30 minutes and gel for 15 minutes. Run at (2 mA/cm2 gel area)/hour.

Enchanced Chemiluminescence (ECL) Reagent

From J Immunol Methods. 2007 Jan 10;318(1-2):11-9 Make the following items as separate solutions:

• 250 mM Luminol in DMSO (442.9 mg in 10 mL DMSO)
• 400 mM 4-iodophenylboronic acid in DMSO (991.3 mg in 10 mL DMSO)
• 1M Tris-HCl, pH 8.8

Store the Luminol and 4-iodophenylboronic acid as 55 µl aliquots and the in the -20°C. To use, add 50 µl Luminol, 50 µl 4-iodophenylboronic acid, and 1 mL 1M Tris-Hcl pH 8.8 to water for a total volume of 10 mL. Luminol will precipitate but will go back into solution quickly with gentle mixing. Add 5.42 µl of 30% H2O2 and mix.

Mild Stripping Buffer for Western Blots (Biorad Formulation)

• 3.75 g Glycine
• 2.15 g Magnesium Acetate Tetrahydrate
• 1.85 g Potassium Chloride
• Distilled water to 450 mL

Dissolve all reagents in the water, pH to 2.2 with HCl. Raise volume to 500 mL. Store at 4°C. Wash blots twice with 10 mL of stripping buffer for ten minutes each, then equilibrate in PBS-T by washing three times with 10 mL for five minutes each.

Plasmid DNA Media (PDM)

From Michael K. Danquah, Gareth M. Forde, Growth Medium Selection and Its Economic Impact on Plasmid DNA Production, Journal of Bioscience and Bioengineering, Volume 104, Issue 6, 2007, Pages 490-497

This media is used for growing E. coli expressing plasmids. It is designed to maximize plasmid output by introducing a buffering system, and is more cost efficient than using LB (by calculating plasmid DNA yield per mL of culture). The media must be filtered sterilized; it cannot be autoclaved.

• 7.9 g tryptone
• 4.4 g yeast extract
• 0.5 g NH4Cl
• 0.5 g MgSO4•7H2O
• 12.8 g Na2HPO4•7H2O
• 3 g KH2PO4

Dissolve reagents in 900 mL ddH2O, top up to 1L, and filter sterilize into 250 mL bottles.

Tony's 4D-6X DNA Loading Buffer for Agarose Gels

This is a modular 6X DNA Load Dye that allows you to control the dyes you want in your load buffer, i.e., if you are concerned about a dye band obscuring your DNA fragment, you can leave it out or swap with another. This is by no means a comprehensive list of dyes; if you have one you like, add it! The recipe was adapted from the following site, where you can also find which dyes comigrate with which DNA of certain sizes: https://openwetware.org/wiki/Agarose_gel_loading_buffer

• 15% w/v Ficoll-400
• 20mM Tris-Cl, pH 8.0
• 60mM EDTA
• 0.48% SDS

Make 50 mL of this solution, divide into five 10 mL conical tubes and add 0.12% Xylene Cyanol to one tube, 0.12% Bromophenol Blue to another tube, 0.48% Orange G to another tube, and 0.12% Cresol Red to the fourth tube, and mix all tubes well. Leave the fifth tube with no dyes added. These are designed such that whatever dyes you want, you mix 4 equal volumes of the respective tubes together in a 1.7 mL tube. For instance, if you want Xylene Cyanol and Orange G, you would add 250 µl of the Xylene Cyanol tube, 250 µl of the Orange G tube, and 500 µl of the colorless tube into a 1.7 mL tube and mix well. If you wanted all the colors, add 250 µl of each colored tube to a 1.7 mL tube and mix well.

100X Denhardt's Solution

For blocking of nylon membranes to be used in northern studies

• 20g Ficoll-400
• 20g Polyvinylpyrrolidone
• 20g Fraction V BSA

Dissolve the solids in 1 L final volume ddH2O. You may add low heat to assist, but if it gets too hot, the BSA will irreversibly precipitate. Filter through a 0.45 µm filter, aliquot 50 mL into a conical tube, and store at -20°C.

10X MOPS Running Buffer for Denaturing Agarose RNA Gels

• 41.8g MOPS (3-(4-Morpholino)propane sulfonic acid)
• 16.6 mL 3M NaOAc
• 20 mL 0.5M EDTA, pH 8.0

Dissolve the reagents in just shy of 1L ddH2O, pH to 7.0 with NaOH, top up to 1L. Store at 4°C protected from light.

Cellprofiler Pipelines

Pipeline for Renaming Channel Files
Pipeline for Correcting DAPI and GFP Fluorescence Channel Threshold Levels
Pipeline for Yeast Nuclear Localization of GFP Tagged Protein

Transcriptional Shutoff Using rpb1-1^TS Saccharomyces cerevisiae Cells

This is our normal protocol for transcriptional shutoffs in cell backgrounds where the RNA Pol II subunit 2b is the rpb1-1^TS mutant allele. These cells transcribe fine at room temp, but raising the temperature to 37°C causes the polymerase to become inactive, allowing a time course analysis of decay (usually by Northern blot and 5' end radiolabeling of transcript-specific complementary ssDNA with T4 PNK and ATP[γ-32P]) after the temperature shift.
Before beginning, make sure to assemble the following items:

• A bucket of crushed dry ice
• A crushed dry ice/methanol bath in a small beaker with a floating tube holder on top
• Time point-labeled two mL microcentrifuge tubes (we like the Big Top brand that hold slightly over 2 mL); standard time course we perform uses 0, 2, 4, 6, 8, 10, 15, 20, 30, and 40 minute time points
• Digital timer capable of counting up, preferably with multiple timers
• Water bath shaker by your bench set to 37°C and containing holders for a 250 mL flask
• Tabletop water bath set to 50°C for warming shutoff media
• A 5 mL micropipetter set to 2 mL and sterile tips for it
• Tabletop centrifuge
• One counter-balance 2 mL tube for the tabletop centrifuge filled with 2 mL of water.
• A vacuum flask assembly for removing supernatant from cells after centrifugation (should have tubing leading to a 1 mL micropipette tip or similar, something that can fit inside a 2 mL tube)

1. Before beginning the initial collection of cells from the media, warm 12.5 mL of the appropriate media containing enough carbon source for a 25 mL culture (i.e., for galactose, 10 mL media and 2.5 mL galactose) in a 250 mL flask to 50°C in the tabletop water bath.
2. Starting from an overnight 200 mL culture in the appropriate selective media, once the culture has reached an O.D.₆₀₀ of 0.4, pour the culture into four 50 mL conical tubes (it is not strictly necessary that the conical tubes be sterile, only clean).
3. Centrifuge at 3,200 x g for 2 minutes.
4. Discard supernatant.
5. Resuspend the cell pellets in 10 mL of total media with no carbon source (resuspend the pellet in one conical tube with the ten mL, move the resuspended cells to the next cell pellet and resuspend that pellet as well, etc., until all four cell pellets have been resuspended in the 10 mL of media).
6. Clear a timer so that it reads 0 (we want to count up).
7. In a quick motion, add the room temperature cells to the 50°C prewarmed media and swirl briefly.
8. Immediately remove 2 mL of this cell suspension with the 5 mL micropipetter and dispense into the first time point tube labeled 0 and place in the tabletop centrifuge and close the lid and turn on.
9. Start the timer.
10. Spin for 15 seconds.
    • While cells are spinning, cap the flask with the remainder of the cells in it and place in the 37°C floor shaker and turn the shaker on.
11. Open the lid of the centrifuge and stop the tubes. Remove the tube with the now pelleted cells and aspirate the supernatant with the vacuum assembly, making sure not to touch the cell pellet.
12. Close the tube lid and place in the dry ice/methanol bath.
13. 15 seconds before the next time point (here, 2 minutes, so 1:45), stop the floor shaker and remove 2 mL from the flask (do not remove the flask from the shaker). Place in the next time point tube, close the lid, and place in the centrifuge and start so that it starts spinning when the timer reads 2 minutes on the nose.
14. Repeat the aspiration of supernatant and putting in the dry /ice methanol bath.
15. Do not forget to turn the floor shaker back on after each time point is collected.
16. Once all the samples have been collected, they can be stored in a -80°C for at most a week before RNA isolation should occur.