Difference between revisions of "Team:DTU-Denmark/Experiments"

Materials

• Fungal plates (either streaked or 3-point)
• Drigalski spatula
• 500 ml shake flasks
• Counting chamber
• Solutions (APB, ATP, PCT, milli-Q, TM)
• 30 °C incubator with shaking
• sterile tea spoon
• mira cloth in funnel (sterile)
• Glucanex
• magnet stirrer
• magnets
• 50 ml sterile falcon tubes
• 0.45 µm filters
• 50 ml syringe
• centrifuge for falcon tubes

Media

• Aspergillus transformation buffer (ATB)
• Aspergillus protoplastation buffer (APB)
• 500 ml shake flasks
• YPD media

Procedure

Initiation

• Streak spore suspension of host strain on YPD plates supplemented with uridine (for this particular fungus) and let grow for a week (there should be black spores!)
• All solutions should be sterile.

Day 1 (inoculation)

• Add 95 ml of YPD media supplemented with uridine to a shake flask and transfer 5 ml of the YPD media to a plate with A. niger. Collect conidia and spores from plate by carefully scraping off the conidia using a drigalski (the spores are hydrophobic and would therefore rather just fly around than actually get into suspension so be careful not to make a mess here). This should give a concentration of around 108 spores/100 ml. It is a good idea to make more than one shake flask at a time.
• Incubate shake flasks at 30 °C, 150 RMP for 48 h.

Day 3 (mycelial harvest)

• Place the sterile funnel with a mira cloth in to a sterile blue cap bottle and transfer the contents of the shake flasks to the mira cloth (content should be brown and thick).
• Wash the mycelia using Aspergillus protoplastation buffer (APB) to remove residual glucose from the mycelia (this can inhibit protoplastation). You need to use quite a bit of APB. squeeze out remaining liquid using a sterile spoon. Then, transfer the mycelium to falcon tubes ( ≈2 g per tube => 1 shake flask ≈ 2 falcon tubes).

Protoplastation

• Add glucanex to APB to get a final concentration of 40 mg glucanex per ml APB and dissolve glucanex via gentle magnetic stirring and no heat.
• Sterile filter 20 ml of APB+glucanex to each falcon tube using a 0.45 µm filter and a 50 ml syringe (there is a bit of resistance in the filter but that's ok).
• Shake/incubate enzyme-mycelium mix at 30 °C, 150 RPM for 2-3 h.
• From now on, whenever you pipette anything with the cells in it, cut of the edge of the pipette tip and CAREFULLY(!) pipette the cells. If you don't do this, they break as they don't have a cell wall to keep them stable.
• Evaluate the number and quality of protoplasts in a microscope and discard a batch that is too diluted (i.e. < 105 protoplasts/ml). Approved protoplast solutions are then diluted by pouring APB up to 40 ml. and the tubess are balanced. Dilute Aspergillus transformation buffer (ATB) to 1/2x with sterile milli-Q H2O and carefully place 5 ml of this on top of the APB, creating an overlay. Centrifuge samples on rotor settings rotor code Sla-600TC; time: 13 min; Speed: 3000g; Temperatire: 16 °C, Acc: 2, Brake: 2 (NB! due to slow acc and brake, this takes forever!)
• In the interphase between the two liquids, a halo of white slurry consisting of concentrated protoplasts should be observed. If there is cell wall debris mixed in with the protoplasts, that's ok. They can still be used. Withdraw the protoplasts with a pipette and wash them in a new falcon tube. Add ATB up to 40 ml and pellet the protoplasts at 3000g for 13 min (acc. 2, brake 2). Discard supernatant by decanting.
• Count protoplasts in microscope by diluting a small sample 1:100.
• Resuspend protoplasts in 4 ml ATB to obtain concentrated solution.

Materials

• DNA
• Protoplasts
• Falcon tubes
• PCT
• TM molten agar
• TM plates
• ATB

Procedure

Regular protocol

• Add at MOST 20 uL DNA ( or max 25% of protoplast volume) and 100 uL of protoplasts in a 50 mL falcon tube.
• Incubate in the falcon tube at RT for at least 30 min.
• Add 1mL of PCT.
• Gently mix by gently swirling the tube in a circular motion – careful they are fragile. Do not vortex or pipette mix.
• Incubate for 5 min at RT.
• Add 3 mL of ATB.
• Add 12 mL of molten (40-45 °C) TM agar.
• Immediately pour mixture directly onto TM plates, and swirl to spread mixture evenly.
• Let the plates settle for a few minutes and incubate at 37 °C for 4 days.

Quick protocol

• Add at MOST 25 uL (1500-5000 ng) DNA ( or max 25% of protoplast volume) and 100 uL of protoplasts in a 2 ml Eppendorf tube. 1 uL (100 ng) pac6 as positive control.
• Add 150 μL PCT with large-nozzle pipette tip.
• Gently mix by swirling – careful the protoplasts are fragile.
• Incubate 10-30 min at room temperature.
• Add 250 μL ATB.
• Distribute transformation mix on osmotic-stabilized selective media and let the agar absorb the mix before incubating

Materials

• biolector

Procedure

• Turn on the machine (bottom in the back, to the right)
• Principal window ----> “Monitor”
• When the lid opens, place the plate in the machine between the metal barrages - it should be firmly secured.
• For create a new program go to second window “Start …” and follow the steps:
• Choose new protocol and give it a name (if it is a warm-up program, specify it - the machine needs to warm up for 20-30 min before you add the plate) ----> Next. Names should be written as DDMMYYiGEM_*temperature*_XXXXX
• Select the type of plate (MTP 48 wells FlowerPlate) ----> Next
• Choose Layout (48 MTP) ----> Next
• Select rpm (1000 rpm), temperature and humidity control, and manual (by defect) ----> Next
• Select the filters to use (Biomass –> Gain 10, PFP –> Gain ?, GFP –> Gain ? ) ----> Next
• Choose cycle time (30 min for the warm-up program and 3 min (more if several filters are addded) for our program) ----> Start
• The machine will ask for refilling the deposit with water. Once done, press OK and the program will start running. Be sure to fill up water once a day.
• When the running is finished, press Stop and save the file in the computer (in the machine, it is saved automatically). You need to press the 'update' button on the computer to get the updated data.
• In the software go to “Data Management” and select “Transfer the data…”. You can create a new folder or select one already created and press create file. The file will be saved in the folder in cvs format.

Materials

• Transformants from whatever assembly method you fashion
• Eppendorf tubes
• Steril toothpicks/inoculation lops/pipette tips for transferring colonies
• Sterile water
• LB media with apropriate antibiotics

Procedure

Preparing the template DNA from the transformants:

• Pick a number of transformants, typically 3-10, from each plate of interest and mark them on the back of the plate.
• Set up 2 eppendorf tubes for each colony and mark them accordingly:
• Fill the first one (1) with 15 µl MQ water.
• The other one (2) remains empty for now.
• Transfer each colony to the eppendorf containing 15 µl water using a sterile toothpick, inoculation loop or autoclaved pipette tip.
• Transfer 5 µl of the water from (1) to the empty (2) tube. This tube (2) is now for safekeeping in case the colony PCR shows that the transformant in question contains the correct insertion.
• Boil the (1) tubes for 10 minutes at 98 °C. Prepare the PCR mastermix, while the colonies are boiling.

Setting up the PCR itself:

• Set up a 25 µl reaction for each colony to be screened, as per NEB'S one-taq PCR protocol (see the protocolfor troubleshooting).

• Run the PCR in the thermocycler (use this website to calculate temperatures used based upon the primers and polymerase used).
• Run the products on a gel to check for correct insertion.
• Prepare an O/N culture from the (2) tubes that have the correct insertions by adding 1 ml LB media to the tube, mixing it and transfer a W-tube containing 4 ml LB media.

Materials

Chemicals

• DpnI enzyme

Procedure

DpnI digestion

• Add 1µL of DpnI to finished 50µL PCR reactions (or .5µL to 25µL reactions). Pipet or invert to mix.
• Incubate the mixture at 37 °C for 1-2 hrs.
  Alternatively, the solution can be left overnight at room temperature. Periodic mixing may aid digestion (but is unnecessary).
• PCR cleanup or gel-purify the reaction for downstream processes OR heat inactivate at 80 °C for 20 min.

Materials

Equipment

• Autoclave
• Freezer
• Centrifuge
• Spektrophotometer

Consumables

• PCR tubes
• 50 mL falcon tubes

Chemicals

• MgCl₂
• CaCl₂
• SOC/SOB
• Glycerol

Procedure

Day 1:

• Autoclave the following:
• Minimum 200 mL 0.1 M MgCl₂
• Minimum 150 mL 0.1 M CaCl₂
• Minimum 100 mL 85 mM CaCl2 + 15% glycerol (v/v)
• Minimum 700 mL SOC
• 3 x shakeflasks (500 mL)
• Freeze at -20 degC (after autoclaving)
• 0.1 M MgCl₂
• 0.1 M CaCl₂
• 85 mM CaCl₂ + 15% glycerol (v/v)
• Minimum 10 x 50 mL falcon tubes
• PCR tubes

Day 2:

• Culture growth
• Early in the morning, start cooling the Sorval centrifuge to 4 C
• Pour 200 mL SOB media into each of the shakeflask (one shakeflask per starter culture).
• Mark the shakeflasks to match the startercultures
• Measure the OD 600 of each starterculture, and inoculate the shakeflask with a volume so the final OD 600 value in the shakeflask culture becomes 0.01
• Grow the shakeflask culture at 37 C with shaking. Measure OD values of the sample every 20 minutes once the OD 600 value is above 0.2.
• When OD 600 is between 0.3 and 0.4, put the cultures into an ice bath immediately, and swirl the shakeflash around in the cold water to cool culture. Chill the culture in the icewater for 20-30 minutes, occasionally swirling the cultures.
• FROM THIS STAGE ON, KEEP CELLS AT ICE/4 °C AT ALL TIMES
• For each shakeflask culture, pour the culture into 3 x 50 mL frosted falcon tubes from the freezer.
• Keep the tubes on ice
• Centrifuge falcon tubes at 3000 x g for 15 min at 4 C (Spin #1 of 4)
• Discard supernatant, and resuspend cells in 15 mL icecold 0.1 M MgCl2
• Keep tubes with cells on ice
• Pool the resuspended cells into one of their matching falcon tubes, so you now have 3 different 50 mL falcon tubes, one with cells corresponding to each of the starter cultures you had.
• Keep tubes on ice
• Centrifuge falcon tubes at 2000 x g for 15 min at 4 o C (Spin #2 of 4)
• Discard the supernatant, and resuspend pellet in 40 mL icecold 0.1 M CaCl2
• Keep tubes on ice
• Let cell suspensions stand in ice for 20-30 minutes
• Centrifuge falcon tubes at 2000 x g for 15 min at 4 C (Spin #3 of 4)
• Discard supernatant, and resuspend pellet in 10 mL icecold 85 mM CaCl 2 + 15 % glycerol
• Keep tubes on ice
• Centrifuge falcon tubes at 1000 x g for 15 minutes at 4 C (Spin #4 of 4)
• Pellet might look small and will be a bit fragile. Handle tubes with care when taking them out of centrifuge
• Attention: The next few steps are best done on ice inside a LAF bench
• Resuspend pellet in 800 uL ice-cold 85 mM CaCl2 + 15 % glycerol
• Put falcon tubes on ice
• Immediately after cells are confirmed resuspended, aliquot 30 uL of the competent cell culture into the chilled PCR tubes
• Put tubes into -80 °C freezer as fast as possible

Materials

• Fungal plates (either streaked or 3-point)
• Drigalski spatula
• 500 ml shake flasks
• Counting chamber
• Solutions (APB, ATP, PCT, milli-Q, TM)
• 30 °C incubator with shaking
• sterile tea spoon
• mira cloth in funnel (sterile)
• Glucanex
• magnet stirrer
• magnets
• 50 ml sterile falcon tubes
• 0.45 µm filters
• 50 ml syringe
• centrifuge for falcon tubes

Media

• Aspergillus transformation buffer (ATB)
• Aspergillus protoplastation buffer (APB)
• 500 ml shake flasks
• YPD media

Procedure

Initiation

• Streak spore suspension of host strain on YPD plates supplemented with uridine (for this particular fungus) and let grow for a week (there should be black spores!)
• All solutions should be sterile.

Day 1 (inoculation)

• Add 95 ml of YPD media supplemented with uridine to a shake flask and transfer 5 ml of the YPD media to a plate with A. niger. Collect conidia and spores from plate by carefully scraping off the conidia using a drigalski (the spores are hydrophobic and would therefore rather just fly around than actually get into suspension so be careful not to make a mess here). This should give a concentration of around 108 spores/100 ml. It is a good idea to make more than one shake flask at a time.
• Incubate shake flasks at 30 °C, 150 RMP for 48 h.

Day 3 (mycelial harvest)

• Place the sterile funnel with a mira cloth in to a sterile blue cap bottle and transfer the contents of the shake flasks to the mira cloth (content should be brown and thick).
• Wash the mycelia using Aspergillus protoplastation buffer (APB) to remove residual glucose from the mycelia (this can inhibit protoplastation). You need to use quite a bit of APB. squeeze out remaining liquid using a sterile spoon. Then, transfer the mycelium to falcon tubes ( ≈2 g per tube => 1 shake flask ≈ 2 falcon tubes).

Protoplastation

• Add glucanex to APB to get a final concentration of 40 mg glucanex per ml APB and dissolve glucanex via gentle magnetic stirring and no heat.
• Sterile filter 20 ml of APB+glucanex to each falcon tube using a 0.45 µm filter and a 50 ml syringe (there is a bit of resistance in the filter but that's ok).
• Shake/incubate enzyme-mycelium mix at 30 °C, 150 RPM for 2-3 h.
• From now on, whenever you pipette anything with the cells in it, cut of the edge of the pipette tip and CAREFULLY(!) pipette the cells. If you don't do this, they break as they don't have a cell wall to keep them stable.
• Evaluate the number and quality of protoplasts in a microscope and discard a batch that is too diluted (i.e. < 105 protoplasts/ml). Approved protoplast solutions are then diluted by pouring APB up to 40 ml. and the tubess are balanced. Dilute Aspergillus transformation buffer (ATB) to 1/2x with sterile milli-Q H2O and carefully place 5 ml of this on top of the APB, creating an overlay. Centrifuge samples on rotor settings rotor code Sla-600TC; time: 13 min; Speed: 3000g; Temperatire: 16 °C, Acc: 2, Brake: 2 (NB! due to slow acc and brake, this takes forever!)
• In the interphase between the two liquids, a halo of white slurry consisting of concentrated protoplasts should be observed. If there is cell wall debris mixed in with the protoplasts, that's ok. They can still be used. Withdraw the protoplasts with a pipette and wash them in a new falcon tube. Add ATB up to 40 ml and pellet the protoplasts at 3000g for 13 min (acc. 2, brake 2). Discard supernatant by decanting.
• Count protoplasts in microscope by diluting a small sample 1:100.
• Resuspend protoplasts in 4 ml ATB to obtain concentrated solution.

Materials

• Fungal plates (either streaked or 3-point)
• Drigalski spatula
• 500 ml shake flasks
• Counting chamber
• Solutions (APB, ATP, PCT, milli-Q, TM)
• 30 °C incubator with shaking
• sterile tea spoon
• mira cloth in funnel (sterile)
• Glucanex
• magnet stirrer
• magnets
• 50 ml sterile falcon tubes
• 0.45 µm filters
• 50 ml syringe
• centrifuge for falcon tubes

Media

• Aspergillus transformation buffer (ATB)
• Aspergillus protoplastation buffer (APB)
• 500 ml shake flasks
• YPD media

Procedure

Initiation

• Streak spore suspension of host strain on YPD plates supplemented with uridine (for this particular fungus) and let grow for a week (there should be black spores!)
• All solutions should be sterile.

Day 1 (inoculation)

• Add 95 ml of YPD media supplemented with uridine to a shake flask and transfer 5 ml of the YPD media to a plate with A. niger. Collect conidia and spores from plate by carefully scraping off the conidia using a drigalski (the spores are hydrophobic and would therefore rather just fly around than actually get into suspension so be careful not to make a mess here). This should give a concentration of around 108 spores/100 ml. It is a good idea to make more than one shake flask at a time.
• Incubate shake flasks at 30 °C, 150 RMP for 48 h.

Day 3 (mycelial harvest)

• Place the sterile funnel with a mira cloth in to a sterile blue cap bottle and transfer the contents of the shake flasks to the mira cloth (content should be brown and thick).
• Wash the mycelia using Aspergillus protoplastation buffer (APB) to remove residual glucose from the mycelia (this can inhibit protoplastation). You need to use quite a bit of APB. squeeze out remaining liquid using a sterile spoon. Then, transfer the mycelium to falcon tubes ( ≈2 g per tube => 1 shake flask ≈ 2 falcon tubes).

Protoplastation

• Add glucanex to APB to get a final concentration of 40 mg glucanex per ml APB and dissolve glucanex via gentle magnetic stirring and no heat.
• Sterile filter 20 ml of APB+glucanex to each falcon tube using a 0.45 µm filter and a 50 ml syringe (there is a bit of resistance in the filter but that's ok).
• Shake/incubate enzyme-mycelium mix at 30 °C, 150 RPM for 2-3 h.
• From now on, whenever you pipette anything with the cells in it, cut of the edge of the pipette tip and CAREFULLY(!) pipette the cells. If you don't do this, they break as they don't have a cell wall to keep them stable.
• Evaluate the number and quality of protoplasts in a microscope and discard a batch that is too diluted (i.e. < 105 protoplasts/ml). Approved protoplast solutions are then diluted by pouring APB up to 40 ml. and the tubess are balanced. Dilute Aspergillus transformation buffer (ATB) to 1/2x with sterile milli-Q H2O and carefully place 5 ml of this on top of the APB, creating an overlay. Centrifuge samples on rotor settings rotor code Sla-600TC; time: 13 min; Speed: 3000g; Temperatire: 16 °C, Acc: 2, Brake: 2 (NB! due to slow acc and brake, this takes forever!)
• In the interphase between the two liquids, a halo of white slurry consisting of concentrated protoplasts should be observed. If there is cell wall debris mixed in with the protoplasts, that's ok. They can still be used. Withdraw the protoplasts with a pipette and wash them in a new falcon tube. Add ATB up to 40 ml and pellet the protoplasts at 3000g for 13 min (acc. 2, brake 2). Discard supernatant by decanting.
• Count protoplasts in microscope by diluting a small sample 1:100.
• Resuspend protoplasts in 4 ml ATB to obtain concentrated solution.