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General microbiology protocols

Protein Purification

Procedure:

LOCATION: MEE LAB Kept at 4ºC — In the MEE fridge Put 30-40ml aliquot of lysis buffer in 50 mL tube on ice in 100 mL beaker Add ? mL of lysis buffer to each cell pellet Turn on large centrifuge and set it to 4ºC (do this now so it can reach this temp when needed) Resuspend via vortex until smooth to avoid clumps of pellet. Very important! Go down Bio superlab on first cloor of KINSC

CHANGE LOCATION: Bio Superlab Put on ear protection! Put up sign on freezer door that indicates sonicator is turned on. Important Saftey Measure Clean sonicator probe using ethanol and dry with Kimwipe before use. Fully immerse sonicator tip in beaker with cell, but make sure the tip does not touch any beaker walls. Sonicator operating settings: Put sonitcater at power level of 40% Operate: 30 seconds on, 30 seconds off. Do for 10 minutes total ( 5 min sonication overall) Spin down in **chilled** ultra-centrifuge Use smaller tubes (these will be located under the swinging bucket centrifuge - specify spatial location later) 14000 RPM for 45 minutes While spinning, start next steps

Column Equilibration

CHANGE LOCATION: MEE LAB Get Nickel agarose high density beads from 4ºC MEE fridge — shake well! To equilibrate the with lysis buffer: Get 15 mL conical and add about 10 mL of lysis buffer (make sure its chiled) Need 4 mL of slurry total. (slurry is the term for a combianation of resin beads and ethanol) 1 mL of resin beads == 15 mg protein (good for our 2 L) Resin is in 50% ethanol, so need 4 mL of resin NOTE: Do not leave out of refrigerator Use spinning bucket centrifuge, 3000 RPM for 2 minutes (be sure to balance properly) - what’s the quick balance method? Discard ethanol Add 2 mL lysis buffer Mix and spin down Carry out Steps d-f a total of 4 times total Remove lysis buffer so that only the resin beads remain (this is to make sure all ethanol is removed)

Gentilly pour cell lysate from Step 12 into a 50 mL tube. Leave 1 mL so that cell debris does not come out (very important step) Add resin beads to the 50 mL tube and use cell lysate to resuspend the resin beads (in its 15 mL tube?) Use cell lysate form 15 mL tubes to resuspend the resin beads Pipette up and down mix all resin into the lysate. Wash walls with lysate to get all resin out. Seal with parafilm. Place flat on orbital shaker for 30 minutes (Good stopping point for lunch)

Disposable Column Get the disposable column and snap off the end. Get the lid and stopper ready.

CHANGE LOCATION: Cold Room in Bio Superlab Work in the cold room in Bio super lab; set up column with clamps holding it in place (example picture) Pour in resin and proteins. Rotate tube to get resin out NOTE: Avoid forming air bubbles Open bottom and let all liquid pour through. Take small volume (2 uL) of flow-through in the Eppendorf tube and measure absorbance. This volume is labelled FLOUGH THOUGH Start Take FLOUGH THOUGH Start to Nanodrop to obtain absorbance readings Th aim is get the column to have strong 260 absorbance due to oligonucleotides: Keep adding lysis buffer until 260 absorbance is zero. This will take about 9-10 volumes NOTE: when adding buffer, use a Pasteur pipette to apply it to the walls of the column so as not to distub the resin Don’t let the resin get dry, keep it hydrated at all times

Measuring Absorbance for Washes Add 200 mL (?) wash buffer Obtain absorbance readings from these washes in the same way that was done for the flowthrough in Steps e-f Th aim is get the column to have strong 260 absorbance due to oligonucleotides: Keep adding lysis buffer until 260 absorbance is zero. This will take about 9-10 volumes

Nanodrop 2 uL "protein" at 280nm, "Overlay spectra" between readouts. Use lysis buffer for blank. Measure blank to makes sure there Nanodrop is clean and that there’s no preexisting absorbance. Note: Looking for strong 260 peak (~0.85)

Goal

To prepare plasmids from a strain of bacteria

Growing Up Strains

1. From glycerol stocks in -20°C freezer, get the strains you want to work with.
   • D39
   • Hermans 930
   • Pmen 4
2. Day 1:
   • Get Blood Plates (1 per strain), inoculation loops, tube holder. Bring everything into the hood.
   • Using an inoculation loop, streak for single colonies onto the entire plate. Obviously change loops between

a) seems like it is roughly 10 ul, your master mix is probably fine.

b) falls a little short of 10 ul, maybe the volume distribution in the 8 tubes was not equal. You can probably proceed.
c) is close to 0 ul, you might have not added the proper volume for all reagents. 
  i) You might have to redo the master mix to avoid an 
  unsuccessful PCR

Procedure:

LOCATION: MEE LAB Kept at 4ºC — In the MEE fridge Put 30-40ml aliquot of lysis buffer in 50 mL tube on ice in 100 mL beaker Add ? mL of lysis buffer to each cell pellet Turn on large centrifuge and set it to 4ºC (do this now so it can reach this temp when needed) Resuspend via vortex until smooth to avoid clumps of pellet. Very important! Go down Bio superlab on first cloor of KINSC

CHANGE LOCATION: Bio Superlab Put on ear protection! Put up sign on freezer door that indicates sonicator is turned on. Important Saftey Measure Clean sonicator probe using ethanol and dry with Kimwipe before use. Fully immerse sonicator tip in beaker with cell, but make sure the tip does not touch any beaker walls. Sonicator operating settings: Put sonitcater at power level of 40% Operate: 30 seconds on, 30 seconds off. Do for 10 minutes total ( 5 min sonication overall) Spin down in **chilled** ultra-centrifuge Use smaller tubes (these will be located under the swinging bucket centrifuge - specify spatial location later) 14000 RPM for 45 minutes While spinning, start next steps

Column Equilibration

CHANGE LOCATION: MEE LAB Get Nickel agarose high density beads from 4ºC MEE fridge — shake well! To equilibrate the with lysis buffer: Get 15 mL conical and add about 10 mL of lysis buffer (make sure its chiled) Need 4 mL of slurry total. (slurry is the term for a combianation of resin beads and ethanol) 1 mL of resin beads == 15 mg protein (good for our 2 L) Resin is in 50% ethanol, so need 4 mL of resin NOTE: Do not leave out of refrigerator Use spinning bucket centrifuge, 3000 RPM for 2 minutes (be sure to balance properly) - what’s the quick balance method? Discard ethanol Add 2 mL lysis buffer Mix and spin down Carry out Steps d-f a total of 4 times total Remove lysis buffer so that only the resin beads remain (this is to make sure all ethanol is removed)

Gentilly pour cell lysate from Step 12 into a 50 mL tube. Leave 1 mL so that cell debris does not come out (very important step) Add resin beads to the 50 mL tube and use cell lysate to resuspend the resin beads (in its 15 mL tube?) Use cell lysate form 15 mL tubes to resuspend the resin beads Pipette up and down mix all resin into the lysate. Wash walls with lysate to get all resin out. Seal with parafilm. Place flat on orbital shaker for 30 minutes (Good stopping point for lunch)

Disposable Column Get the disposable column and snap off the end. Get the lid and stopper ready.

CHANGE LOCATION: Cold Room in Bio Superlab Work in the cold room in Bio super lab; set up column with clamps holding it in place (example picture) Pour in resin and proteins. Rotate tube to get resin out NOTE: Avoid forming air bubbles Open bottom and let all liquid pour through. Take small volume (2 uL) of flow-through in the Eppendorf tube and measure absorbance. This volume is labelled FLOUGH THOUGH Start Take FLOUGH THOUGH Start to Nanodrop to obtain absorbance readings Th aim is get the column to have strong 260 absorbance due to oligonucleotides: Keep adding lysis buffer until 260 absorbance is zero. This will take about 9-10 volumes NOTE: when adding buffer, use a Pasteur pipette to apply it to the walls of the column so as not to distub the resin Don’t let the resin get dry, keep it hydrated at all times

Measuring Absorbance for Washes Add 200 mL (?) wash buffer Obtain absorbance readings from these washes in the same way that was done for the flowthrough in Steps e-f Th aim is get the column to have strong 260 absorbance due to oligonucleotides: Keep adding lysis buffer until 260 absorbance is zero. This will take about 9-10 volumes

Nanodrop 2 uL "protein" at 280nm, "Overlay spectra" between readouts. Use lysis buffer for blank. Measure blank to makes sure there Nanodrop is clean and that there’s no preexisting absorbance. Note: Looking for strong 260 peak (~0.85)

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