KU ISTANBUL WIKI - Template

Protocols

SOC medium recipe

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

Note: add Glucose after autoclaving the solution with the remaining ingredients, and letting it cool down. Sterilize the final solution by passing it through a 0.2 µm filter.

LB Broth Protocol

10 gr peptone
5 gr yeast extract
10 gr NaCl
up to 1000ml dH20 & autoclave

LB Agar Protocol:

10 gr peptone
5 gr yeast extract
10 gr NaCl
15 gr agar
up to 1000ml dH20 & autoclave

Addition of antibiotics to LB medium and their concentrations

Ampicillin 100 µg/mL
Kanamycin 50 µg/mL

Cell Lysis Protocol

Preparation of lysis buffer

50 mM Tris pH 7.5
300 mM NaCl
5% v/v Glycerol supplemented with 0.01% Triton X-100

Soak cells in the lysis buffer
Sonication with Branson W250 sonifier
Centrifugation by Ti45 rotor (Beckman) at at 40000 rpm for 30 minutes at 4°C
Pellets remaining in the bottom of the tubes are discarded and the clear supernatant is ready for nickel chromatography.

His tag Purification protocol

https://cdn.cytivalifesciences.com/dmm3bwsv3/AssetStream.aspx?mediaformatid=10061&destinationid=10016&assetid=13161

SDS-Page Protocol IGEM team

https://www.bio-rad.com/webroot/web/pdf/lsr/literature/Bulletin_6040.pdf

Transformation(BL21 Competent Cells)[TuDelft2016iGEM]

Put a tube of BL21 Competent E. coli cells on ice for 10 minutes.
Add 2µL of plasmid DNA to the cell mixture.
Mix by flicking the tube.
Place the tube on ice for 30 minutes.
Heat shock at 42ºC for exactly 10 seconds.
Place on ice for 5 minutes.
Add 950µL of SOC medium at room temperature.
Incubate at 37ºC with shaking for 1 hour.

NOTE: Not at the thermoblock, use a shaking incubator.

To increase the concentration of cells before plating, centrifuge the tubes at 4000rpm for 3 minutes, remove 700µL of medium, resuspend the pellet in the remaining volume.
Plate 70µL of the transformation on selective LB plates and incubate at 37ºC overnight.

Overnight Cell Cultures[TuDelft2016iGEM][Modified]

Prepare 20mL of selective LB with Kanamycin per colony to be picked.

NOTE: You can use a sterile bottle to prepare the total volume.

Transfer 10mL of selective LB to a 50mL Falcon tube. Prepare 2 tubes per colony: 1 for miniprep and 1 for cryostocks.
Pick 1 colony using an inoculation loop and soak it in the LB. Make sure you have cells left for the second tube.
Take the tubes to room F1.690 and incubate overnight at 37ºC in the shaking incubator.

Gibson Assembly Protocol (will be prepared)

If homemade master mix aliquots are available, and less than 1 year old:

5µL aliquots or a 2X stock of Gibson master mix. Tubes are in both a 96 well holder, and a 50mL tube.
Sterile, nuclease-free water
Positive control: Positive Control DNA Mix (see below)

Use PCR to produce the DNA segments needed for assembling the new construct. Generally, it is best to use a high fidelity polymerase, such as Phusion, to amplify your Gibson fragments. See our following protocol for setting up a standard PCR reaction
Confirm the success of each PCR by running 5µL of the reaction on an agarose gel. Gel purifying your fragments is always better than PCR purifying them - even if you only observe a single band on your gel.
Mix 10ng-100ng of each of your DNA fragments together (such that their ratios are equimolar) into a 5µL total volume. Therefore, the length of each fragment, and the concentration of the fragments must be taken into account. Attached at the bottom of this page is an excel spread sheet calculator for an easy and accurate calculation of the amount of DNA needed.
If using the 2X Gibson Master Mix from NEB, add 10µL of total DNA (containing all of your fragments) to 10µL of mix. If using the homemade Gibson mix (recipe at the bottom of this page), add 5µL of DNA to 15µL of mix. Be careful with pipetting small volumes.
Mix well by pipetting.
Incubate the reaction at 50°C for 1 hour.
We generally use chemically competent cells for transforming Gibson reactions. For each Gibson reaction, you will do two transformations:

Perform a 1:4 dilution of your Gibson reaction in nuclease-free water, add 4µL to 50µL of chemically competent cells
Add 4µL of your Gibson reaction to 50µL competent cells without diluting
Note = If electroporating, dilute the reaction 1:5 in water or you can PCR purify the reaction prior to transformation.

Follow our transformation protocol for chemically competent cells
Run an aliquot of your final reaction on a gel to verify the presence of your construct. It may be useful to make a 'no incubation' control to determine if the chemistry of the reaction happened.

The Positive Control DNA Mix for Gibson Assembly consists of a two-piece assembly of pUC19. It is designed such that 5uL of the Positive Control DNA Mix is to be added to 15uL of Gibson Assembly Master Mix along side experimental reactions. Both pUC19 segments are between 1.3kb and 1.4kb in size. To construct the positive control reaction mix:

PCR amplify the two pUC19 fragments - fragment 1 (F1) and fragment 2 (F2).

Use primers pUC19 F1 Gib FW (5'-CTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGG-3') and pUC19 F1 Gib RV (5'-ACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAA-3') to create F1 and pUC19 F2 Gib FW (5'-TTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGT-3') and pUC19 F2 Gib RV (5'-CCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAG-3').
Use the following reaction mixture:

32.7µL dH2O
10µL 5X HF Buffer
1µL 10uM dNTP
2.5µL 10uM FW primer
2.5µL 10uM RV primer
1µL ~3ng/µL pUC19 template
0.3µL phusion polymerase

Use the following PCR program for both F1 and F2 PCRs:

98°C - 0:30
98°C - 0:10
58°C - 0:30
72°C - 0:45
Go to step 2, 25X
72°C - 10:00

4) After the reaction completes, add 1µL of Dpn1 (20,000 U/mL) to each reaction, briefly vortex and spin down the reactions, and then incubate in the PCR machine at 37°C for 30 minutes. To test the effectiveness of your Dpn1, construct the above PCR reaction without Phusion polymerase, digest and purify it in parallel with the F1 and F2 PCRs, and transform it along side your other reactions. Any ampicillan-resistant transformants produced from from transforming this negative reaction indicate your Dpn1 digest was ineffective at digesting the pUC19 template. The solution to this is to use fresh Dpn1 in your digest, or gel extract F1 and F2 instead of using a Dpn1 digest and column purification to remove pUC19 template.

5) Test the success of the PCR by gel electrophoresis and purify the reaction using a PCR cleanup kit, and elute with buffer or water without EDTA. Quantify the concentration of F1 and F2 purifications.

Combine F1 and F2 PCR products to a final concentration of 2.8ng/µL of each fragment. Dilute with the elution buffer used in the PCR purification if needed. This final mixture is the Positive Control DNA Mix.

Using 5µL of this reaction provides approximately 1ng/100bp of each fragment in the Gibson Assembly reaction.

Gibson_Master_Mix_Gibson_2011.xlsx

Bens_Gibson_Assembly.xlsx

https://barricklab.org/twiki/bin/view/Lab/ProtocolsGibsonCloning#:~:text=Gibson%20Cloning%20is%20a%20technique,isothermal%20assembly%20of%20overlapping%20dsDNA

PCR Protocol

https://www.cytivalifesciences.com/en/us/shop/chromatography/resins/affinity-tagged-protein/histrap-hp-histidine-tagged-protein-purification-columns-p-00250

Competent Cell Preparation by Calcium Chloride Protocol [MetuHSiGEM2016]

Inoculate 1ml ONC to 100 ml LB in a flask.
Incubate the flask for 2-4h at 37 ˚C, check via spectrophotometer at 600nm for 0.300..
Divide the solution into two falcon tubes(50 ml).
Spin them down at +4 ˚C and 5000g for 5min.
Discard supernatant.
Resuspend the cells with 10 ml cold CaCl2.
Put in ice for 10 minutes.
Spin the suspension down at +4 ˚C, 5000g for 5 minutes.
Discard supernatant.
Add 10 ml cold CaCl2 and resuspend the cells.
Put the cells in ice for 30 min.
Centrifuge at 5000 rpm for 5 min at +4 ˚C.
Discard supernatant.
Put 1 ml CaCl2 and dissolve the pellet.
Put it on ice for 5 min. and keep it at +4 ˚C

Polysilicate layer in silicatein expressing cells[TuDelftiGEM2016]

Inoculate a fresh culture of cells from an overnight culture. Make a dilution of around 1/100 of the overnight culture into fresh selective LB.
Incubate in the shaking incubator at 37ºC and 250 rpm until it reaches exponential phase (just measure OD600 from time to time until duplication time is close to 20 min).
Induce the culture with 1/1000 volume of IPTG 1M.
Incubate in the shaking incubator at 37ºC and 250 rpm for 3h.
Add sodium silicate to a final concentration of 60µM from the 1mM stock (maybe new stock needs to be made from the 1M stock, keep sterile!!).
Incubate at 37ºC and 250 rpm for 3-4h, after this the cells are ready for further experiments.

Growth Study[TuDelftiGEM2016]

Follow the Polysilicate layer protocol until step 5.
Take a 100µL sample directly after adding the sodium silicate, this is t=0.
Incubate at 37ºC and 250 rpm and take a 100µL sample every hour.

For each sample:

Make a series of dilutions in 1/10 steps until reaching a 1/106. It can be done in a 96 well plate by adding 10µL of the previous dilution in 90µL of LB medium.
Plate in agar plates 20µL of dilutions 1/100, 1/103, 1/104, 1/105, 1/106.

Incubate at 37ºC overnight.
Hopefully, in some of the dilutions the colonies will be countable. Count colonies in plates for at least one dilution of each strain and time point to make a growth/viability curve.

Fluorophores Kinetic Cycle[TuDelftiGEM2016][Will Be Modified]

Grow cells expressing the fluorophores of interest in eM9 medium since LB has its own fluorescence.
Measure OD600 overnight cultures.
Dilute in eM9 medium an aliquot of each culture to an OD600 of 0.1.
Add 100µL of diluted culture to 4 wells per culture (that way we obtain 4 replicas of the experiment), and also make blanks with sterile eM9.
Program the plate reading machine so it incubates the plate at 37ºC with shaking and makes an OD600 and fluorescence measurement every 15min for 24h.

Excitation and emission wavelengths to be used for each fluorophore are:

EGFP: ex:488 em:507
Emerald: ex: 487 em: 509
mApple: ex: 568 em: 592
Superfolder GFP: ex:485 em:510
TagBFP ex:402 em:457
TagGFP ex: 482 em: 505
TurboGFP ex:482 em: 505
mStrawberry ex: 574 em: 596

Silicic Acid Test

For a 1 mL sample:

Add 20µL of reagent Si-1 and mix.
Wait for 3 min.
Add 20µL of reagent Si-2 and 100µL of Si-3 and mix.
Wait for 10 min.
Measure absorbance at 824nm.

Rhodamine123 staining[TuDelfiGEM2016]

Add 1/1000 volume of Rhodamine123 to the culture to be stained.
Wait for 10 min.
Centrifuge for 1 min at maximum speed.
Wash the cells 5 times with PBS.
Prepare samples for microscopy in a glass slide.
Rhodamine123 is a fluorescent dye which is excited at 546nm and emits
green fluorescence ( Li, Chu & Lee, 1989 )( Johnson, Walsh & Chen, 1980 )

UV Sterilization[TuDelftiGEM2016]

Plate 30µL of cells on selective LB plates.

Positive control: dead culture (for example, autoclaved).
Negative control: culture of living cells.
Test: culture of living cells.

Expose the test plates to UV light using the Stratagene UV Stratalinker 1800 at power 120000µJ and 240000µJ, respectively.
Incubate all plates at 37ºC overnight.

SEM Sample Preparation[TuDelftiGEM2016][ZEISS EVO LS15 SCANNING ELECTRON MICROSCOPE]

Centrifuge 1mL of culture for 1min at maximum speed.
Discard the supernatant
Resuspend the pellet with 3% glutaraldehyde solution. NOTE: Glutaraldehyde is a dangerous chemical, wear gloves and safety goggles!
Let it stand for 10min.
Centrifuge for 1 min at maximum speed.
Discard the supernatant.
Resuspend the pellet in a 30% ethanol solution.
Centrifuge for 1 min at maximum speed.
Discard the supernatant.
Repeat steps 7-9 with ethanol solutions of 50%, 70%, 80%, 90%, 96% and 100%.
Resuspend the supernatant one last time in 100% ethanol.
The samples are ready to be put in a glass slide

YEAST Transformation (according to S. Elledge)

Note: While making competent cells don’t put cells on ice at any stage or use cold centrifuges. To freeze and thaw, cells go from room temperature straight into the freezer and vice-versa.

Solutions:

LiSorb: 100 mM Lithium acetate (LiOAc), 10 mM Tris pH 8, 1 mM EDTA pH 8, 1 M Sorbitol (use sorbitol for molecular biology from Merck). Filter sterilise and keep at room temperature

LiPEG: 100 mM LiOAc, 10 mM Tris pH 8, 1 mM EDTA pH 8, 40% PEG3350. Filter sterilise and keep at 4°C.

Making competent cells:

1. Inoculate cells the day before in 50 ml of appropriate medium and incubate at 30°C or 23°C (temperature sensitive mutants).

2. Measure OD600 and dilute culture in 50 ml medium to OD600 of about 0.15.

3. Incubate until OD600 reaches 0.8 – 1.0 (approx. 2 x 107 cells/ml).

4. Transfer to 50 ml Falcon tubes and centrifuge (3200 rpm, 2 min, room temperature).

5. Discard supernatant and resuspend cell pellet in 45 ml sterile bidest. Water.

6. Centrifuge as before. Discard supernatant and resuspend cell pellet in 20-25 ml LiSorb.

7. Centrifuge as before. Discard supernatant and centrifuge again to get rid of the excess of LiSorb.

8. Resuspend the pellet of 1 OD 50 ml culture in 300 µl LiSorb and 50 µl denatured salmon sperm DNA (If cell amount is different scale up or down accordingly. I.e. if you had 0.8 OD 50 mL culture use 240 µl (300 X 0.8) LiSorb and 40µl (50 X 0.8) denatured salmon sperm DNA.

9. Make aliquots of 100 µl and put straight into the -80°C freezer.

Making denatured Salmon Sperm DNA (Do this step before hand)

To denature the salmon sperm DNA (carrier DNA): heat up salmon sperm DNA for 5 min at 95°C and cool down on ice for 5 min. Keep on ice until usage, freeze the remaining DNA and you can reuse the frozen one after denaturing again).

Transformation:

Use fresh cells or thawed frozen competent cells (with carrier DNA). Do not keep cells on ice. Keep cells at room temperature (RT).

Use 50 µl of competent cells per transformation. For cassette integration, use 5 µl of PCR product (considering that the PCR band is 3x stronger than the marker). For integration using plasmids, use 2-5 µl of 3-5 µg plasmid digested with the appropriate enzyme in 50 µl). For plasmid transformation, use 1-3 µl of DNA.

Don’t forget to do always a “NO DNA control”.

1. Add DNA to competent cells, mix well and incubate for 15 min at RT (incubation time may be skipped if doing plasmid transformation but it is essential if integration into the genome is required).

2. Add 300 µl of LiPEG, vortex well and incubate for 15-20 min at RT.

3. Add 35 µl of supernatant by aspiration.

6. Resuspend cells according to DMSO, vortex and incubate straightaway for 15 min in the 42oC water bath. If transforming temperature sensitive mutants, reduce the incubation time at 42°C to 12 min.

5. Centrifuge for 2-3 min at 3000 rpm, remove the sgly as following:

For auxotrophies, resuspend the cells in 200 µl of PBS and plate out onto appropriate selection plates.

For KanMX6 cassettes, resuspend the cells in 1 ml of YPDA and incubate for 3-4 h at 30oC or 4-5 h or o/n at 23°C. Spin down cells (3200 rpm 2 min). Remove 800 µl medium. Resuspend cells in residual 200 µl. Plate out onto selection plate
For hygromycin and natrunculin cassettes, incubate for 6 h-12h at 30°C or overnight at 23°C. Spin down cells. Remove 800 µl medium. Resuspend cells in residual 200 µl. Plate out onto the selection plate.

Notes: Kanamycin (G418) cassettes give a high noise background growth. To identify the true positive transformants, replica plate transformants onto fresh G418 plates 1 day after the transformation. Only colonies that have integrated Kan cassettes will grow on the replica plate.

Plates and Media

Always Work under sterile conditions! Use Bunsen burner or laminar flow hood when opening bottles!

SC-X Medium (Filter sterilised OR autoclaved) (X= URA or LEU or HIS or TRP)

SC-X WITH GLUCOSE

For 500mL Medium:

Bacto yeast nitrogen base without amino acids 3.4 g
SC-X drop out mix 1 g
Glucose 10 g
Adenine 0.05 g
Bi-dest. water up to 500 ml

Note: Stir well until dissolved. Sterilise by filtration or autoclave

SC-X / Raf Medium (Filter sterilised) (X= URA or LEU or HIS or TRP)

SC-X WITH RAFFINOSE ONLY

For 500mL Medium:

Bacto yeast nitrogen base without amino acids 3.4 g
SC-X drop out mix 1 g
Raffinose 15 g
Adenine 0.05 g
Bi-dest. water up to 500 ml

Note: Stir well until dissolved. Sterilise by filtration. Don’t autoclave! Raffinose can not be autoclaved!

SC-X / RAF GAL Medium (Filter sterilised) (X= URA or LEU or HIS or TRP)

SC-X WITH RAFFINOSE AND GALACTOSE

For 500mL Medium:

Bacto yeast nitrogen base without amino acids 3.4 g
SC-X drop out mix 1 g
Raffinose 15 g
Galactose 10 g
Adenine 0.05 g
Bi-dest. water up to 500 ml

Note: Stir well until dissolved. Sterilise by filtration.Don’t autoclave! Raffinose and Galactose can not be autoclaved!

SC-X Agar Plates (WITH GLUCOSE) (X= URA or LEU or HIS or TRP)

For 1 Liter Agar:

Mix A: (Use a 1 liter or 500mL bottle)

Bacto yeast nitrogen base without amino acids 6,7 g
SC-X drop out mix 2 g
Glucose 20 g
Bi-dest. water up to 500 ml

Note: Stir well until dissolved and autoclave

Mix B: (Use a 1 liter bottle with a magnetic stir-bar)

Bacto Agar 20 g
Bi-dest water up to 500 ml

Note: Add one magnetic stir-bar to the bottle and autoclave.

1. After autoclaving, carefully add the Mix A to the Mix B (the Agar containing bottle), never the other way round, when both solutions are still hot. Add mix B slowly to the side of the bottle to avoid formation of bubbles.

2. Stir well. Pour into plates (~20 ml per plate) under the hood or near the Bunsen burner.

SC-X RAF/GAL Agar plates (with RAF/GAL) (X= URA or LEU or HIS or TRP)

For 1 liter Agar:

Mix A: (Use a 1 liter or 500mL bottle)

Bacto yeast nitrogen base without amino acids 6.7 g
SC-X drop out mix 2 g
Raffinose 30 g
Galactose 20 g
Bi-dest. water up to 500 ml

Note: Stir well until dissolved. Filter sterilise. Don’t autoclave!

Mix B: (Use a 1 liter bottle with a magnetic stir-bar)

Bacto Agar 20 g
Bi-dest. Water 500 ml

Note: Add one magnetic stir-bar to the bottle and autoclave.

1. Take out of the autoclave and add mix B (see below) while still hot (70-80°C).

2. Carefully add the Mix A to the Mix B (the Agar containing bottle), never the other way round! Add mix B slowly to the side of the bottle to avoid formation of bubbles.

3. Stir well. Pour into plates (~20 ml per plate) under the hood or near the Bunsen burner.

YPAD medium

For 500 mL:

Bacto Yeast Extract 5g
Bacto Peptone 10 g
Glucose 10 g
Adenine 0.05 g
Bi-dest. Water up to 500 ml

Stir well with stir-bar until dissolved.
Autoclave or filter sterilize depending on your purpose.

YP/RAF MEDIUM

For 500 mL:

Bacto Yeast extract 5 g
Bacto Peptone 10 g
Raffinose 15 g
Adenine 0.05 g
Bi-dest. Water up to 500 ml

Stir well with stir-bar until dissolved.
Filter sterilize! Raffinose cannot be autoclaved!

YP/RAF/GAL MEDIUM

For 500 mL:

Bacto Yeast extract 5 g
Bacto Peptone 10 g
Raffinose 15 g
Galactose 10 g
Adenine 0.05 g
Bi-dest. Water up to 500 ml

Stir well with stir-bar until dissolved.
Filter sterilize! Raffinose and Galactose cannot be autoclaved!

YPD Agar

For 1 liter Agar:

1. Prepare the following solution (YPD solution) in a beaker:

Bacto Yeast Extract 10 g
Bacto Peptone 20 g
Glucose 20 g
Bi-dest Water up to 1000 ml

Note: Stir well with stir-bar until dissolved.

2. Add following to a 1 liter bottle:

Bacto Agar 20 g
one magnetic stir-bar:
1000mL YPD solution

Autoclave.
Pour into plates.

YP RAF/GAL PLATES

For 1 liter YEB Agar:

1. Prepare the following solution (YEB solution):

Bacto Yeast extract 10 g
Bacto Peptone 20 g
Bi-dest. Water up to 700 ml

Note: Stir well with stir-bar until dissolved.

2. In a 1 liter bottle add followings:

Bacto Agar 20 g
YEB Solution 1000mL
Magnetic stir-bar

Note: Autoclave to obtain YEB Agar! Do not let the agar cool after autoclave! Add immediately the Raf/Gal solution

RAF/GAL Solution

In a separate beaker prepare the following solution:

Raffinose 30 g
Galactose 20 g
Bi-dest. Water up to 300 mL

Notes:

Stir well with stir-bar until dissolved.

Filter sterilize! Raffinose and Galactose cannot be autoclaved!
Keep the Raf/Gal solution at 42°C until the YEB Agar is autoclaved!

MIXING YEB AGAR With RAF/GAL Solution

1. Take YEB AGAR out of the autoclave and add mix with RAF/GAL solution (see below) while the agar is still hot (70-80°C).

2. Carefully add the RAF/GAL solution to the YEB AGAR, never the other way round! Add RAF/GAL solution slowly to the side of the bottle to avoid formation of bubbles.

3. Stir well. Pour into plates (~20 ml per plate) under the hood or near the Bunsen burner.

SC-X Agar with Antibiotics

Yeast Nitrogen Base (Difco) w/o amino acids and w/o ammonium sulfate 1,7 g/L
Monosodium Glutamic Acid 1 g/L
SC-X Drop out mix 2 g/L
Glucose 20 g/L
Agar 20 g/L

Notes:

Autoclave agar separately from others (similar to other SC-X plates)
add standard concentration of antibiotics
Pour into plates

Yeast Expression and Selection Protocol

Plant yeasts in SC minus His and Leu + Raffinose/Galactose Medium.
Grow yeast in SC minus His and Leu and we will add galactose in the wanted time point to start a powerful expression.

Yeast Strains

MGY96 expresses GST-Cdc5 deltaN70 S165D, T238D
MGY97 expresses GST-Cdc5 deltaN70 N209A (GST-Cdc5KD)

Note: Expression of both is induced with galactose

Induction

Day 1. Pick a colony into 10ml of liquid YEP medium plus 1% glucose and grow overnight at 30oC to stationary phase.
Day 2. About 5pm, make a 1:1500 dilution into fresh YEP medium with 1% sucrose as carbon source and grow for 16h overnight. (You are aiming for 5 X 10 7 cells per ml after overnight growth)
Day 3. At about 9am the culture should be in log phase at about 5 X 107 ml. Dilute 3 fold with YEP+1.33% galactose to give a final concentration of 1% galactose, and grow a further 6-9h to induce recombinant protein expression. Cell pellets are harvested by centrifugation, snap frozen, and stored at -80oC. Initially, 50ml induction cultures are sufficient to test for expression of recombinant protein. Afterwards culture volumes can be scaled up as required.

Notes:

Don’t try to use galactose to induce with medium that already contains large amounts of glucose – it won’t work as glucose represses the Ga11-10 promoter even when galactose is present. Have YEP made up without an added carbon source, this way you can control what is happening. The little bit of glucose in the inoculum from the Day 1 culture is all gone by the morning of Day3.

You can use cheaper galactose such as that produced by Acros Organics (cat no 150610010)

Overnight induction did not work for us.

Cell lysis and recombinant protein purification.

GST Breakage buffer:

50mM Tris pH 7.5
250 mM NaCl
4mM DTT
5mM EDTA
10mM NaF
10mM beta glycerophosphate
1 mM Na orthovanadate
1 pill/50ml Roche complete protease inhibitor
10% glycerol
0.5-1% NP 40 (Nonidet)
1mM PMSF

GST Wash buffer:

50mM Tris pH 7.5
250 mM NaCl
1mM DTT

GST elution buffer:

50mM Tris pH 7.5
250 mM NaCl
0.1% Triton
20mM reduced glutathione

Three methods of cell lysis:

Small amounts of cells are used for ‘protein minipreps’. 200-300mg of induced cells obtained from 25-50ml of induced culture are lysed by shaking with glass beads in a Ribolyser machine (Hybaid). Unfortunately, Hybaid no longer produces this machine, we are looking for an alternative to recommend.
Larger amounts of cells are lysed in a Bead Beater (Biospec Products, Bartlesville, Oklahoma, USA).
Larger amounts of cells are lysed in a French pressure cell.

Protein minipreps

For protein ‘minipreps’ up to 300 mg of cells from 50 ml test cultures of induced cells are resuspended in 0.5ml breakage buffer and 0.5mm dia glass beads (Biospec Products, Bartlesville, Oklahoma, USA) are added to the meniscus in a 2ml Ribolyser tube. Cells are lysed by three 10 sec treatments on a Ribolyser machine separated by 1 min intervals on ice to avoid overheating. To recover the lysates, the base of the Ribolyser tube is pierced with a hot needle and the tube is put into a 15 ml tapered plastic centrifuge tube which already contains a 1.5 ml microfuge tube. This assembly is centrifuged briefly on a bench centrifuge to recover the lysate into the lower microfuge tube. Particulates are removed by two 10 min centrifugations at 13K in a refrigerated microfuge. Note, with larger amounts of cells, yields are improved by recovering the pellet of the first clearing spin and subjecting it to a second bout of Ribolyser treatment to complete cell lysis. The cleared lysate is added to glutathione-sepharose or nickel sepharose beads for affinity chromatography purification. 50-100μl of bead slurry are sufficient for an individual protein miniprep. Separation of eluted material from the sepharose matrix is most easily performed using devices such as an Amicon centrifugal filter.

Larger protein preps

Larger quantities of cells are lysed in a French pressure cell or, preferably, in a Bead Beater (Biospec Products, Bartlesville, Oklahoma, USA). Cells are lysed in a minimum of 5ml breakage buffer /1g cells. We usually pass cells twice through a French pressure cell. With the Bead Beater, we use a 50:50 mixture of cell suspension and 0.5mm diameter beads for breakage with the breakage chamber covered with an ice + salt mix. The cells are broken by 5 x 30 sec treatments interspersed with 5min cooling periods. Cleared lysates are prepared by two 30 min clearing spins at 30000g. After 2h binding the glutathione or nickel sepharose, the affinity purification beads are packed into a small column for washing and elution.

Proteins produced are analysed by electrophoresis through SDS-polyacrylamide gels and visualised by staining with Coomassie blue.

Notes:

The binding and elution steps are essential for evaluation experiments as it is not usual to see an induced protein in total cell lysates.
Affinity purified protein should be visible by staining. If you need to detect products by Western blotting, something is wrong. In fact, when there is reasonable expression it is possible to see purified protein bands in unstained gels. After removing one of the glass plates, you need to look obliquely across the dry surface of the gel to see a dimple in the surface of the gel where the band is.

Yeast Expression Protocol

https://edisciplinas.usp.br/pluginfile.php/4613819/mod_resource/content/1/yeastnielsen2014.pdf

Synthetic dye insertion protocol

(This is taken from another paper)

HeLa, NIH3T3, HEK293 and EL4 cell lines were used in this study.

1. Cells were grown at 37 °C with 5% CO2 in full growth medium (DMEM medium supplemented with 10% fetal bovine serum and 1% pen-strep).

2. Cells were washed with PBS, then trypsinized (except for EL4) and transferred to serum free growth medium containing a fluorescent dye.

The following dyes were used: 100 µM Calcein-AM (Life Technologies), 1 mM fluorescein sodium salt (Sigma-Aldrich), 3 mM Pyrromethene 556 (Exciton Inc), 1 mM Rhodamine 6G (Sigma-Aldrich) and 2 wt% Fluorescein isothiocyanate-dextran (FITC-dextran, MW 2,000,000, Sigma-Aldrich).

3. The cells were incubated with the dye solution at standard growth conditions for 30 min and then transferred into the space between the mirrors.

For FITCdextran the cell dispersion was used immediately without incubation.

Red blood cells were collected from a mouse according to protocols in compliance with institutional guidelines and approved by the Institutional Animal Care and Use Committee (IACUC) at the Harvard Medical School, and diluted 1:10 with PBS containing 1mM fluorescein sodium salt dye.

(This is my original protocol for RBCs) (Fatih)

Protocol 1 (RBC isolation from the whole blood):

1. 300 μL of Citrate Phosphate Dextrose Solution (CPD) is taken into the Eppendorf tube.

2. 100 μL of whole blood is taken by pricking a healthy volunteer’s finger. Blood is taken using a micropipette and immediately transferred into CPD solution for anticoagulation.

3. CPD and blood are mixed by shaking the tube gently.

4. The mixture is centrifuged at 1000 g for 10 min.

5. The upper volume of the mixture (white blood cells and plasma) is removed.

6. 300 μL of PBS is added onto pellet and centrifuged at 1000 g and 400 g for 10 min.

7. Steps 5 and 6 are repeated 2 more times.

8. 300 μL of PBS is added onto pellet (after 4th centrifugation).

Protocol 2 (loading RBCs with SPIONs):

(we can use this protocol for synthetic dyes, SPION=magnetic nanoparticles, molecule sizes are around 100nm)

1. Following sample is prepared:

10 μL of RBC suspension (from the stock prepared in protocol 1, step 8) + 80 μL of 0.4% NaCl suspension (prepared with distilled water) + 10 μL of SPION from main stock (taken after thoroughly vortexed).

2. The sample is placed into a box full of ice (4 °C), and the ice box is placed onto a shaker and incubated for 1 hour.

3. After incubation at 4 °C, 500 μL of PBS is added onto the sample and incubated at 37 °C for 1 hour.

4. After incubation at 37 °C, sample is centrifuged at 400 g for 10 min.

5. After centrifugation, the upper volume of the mixture is removed.

6. 300 μL of PBS is added onto the pellet and centrifuged at 400 g for 10 min.

7. Steps 5 and 6 are repeated two more times.

8. 300 μL of PBS is added onto the pellet (after 4th centrifugation).

9. Sample is placed into 4 °C for later use.

Please look for this protocol too for synthetic dye insertion:

doi.org/10.1002/0471142735.ima03bs111

DAPI STAINING YEAST

DAPI staining yeast

1mL yeast + 0.1mL 36% formaldehyde. 1-2H/Rt
Wash 2x 1mL H2O (Spin at 10K, 5" ok). May leave at 4oC
To stain: Spind down and resuspend in 300 ul H2O. Add 700 ul 100% EtOH. Leave for 30 min/Rt.
Spin down, resuspend 500 ul H2O and sonicate 5 sec.
Spin down (10K, 5") and remove top 300 ul. Vortex.
To see, mix 4 ul cells and 4 ul Eileen's premade DAPI + mounting media on slide. Microscope immediately or seal with nail polish & keep dark. **Must seal if you want to take pictures.
EILEEN'S Premade DAPI/mounting media:

•50mg p-phenylenediamine (Sigma in 5mL 1X PBS; adjust to pH 9.0 (NaOH)

•add 45mL glycerol & stir to homogeneity

•add 2.25 ul 1mg/ml DAPI. Store -70oC/dark

[ ] f 45ug/ml DAPI

use 1:1 to stain cells, ie 4 ul cells + 4 ul DAPI

•Alt: Sonicate cells, mix in triton x100 (to 1% final) & seal immediately!

Team:KU ISTANBUL/Protocols

Protocols