Protein+Production+in+Plate+Format

Protein Production in Plate Format Miscellaneous Notes About Protein Production

Materials Note that this spreadsheet could potentially be modified so that some of the Biomek tips are reused rather than new ones used for each step. In order to do this, which tip box is used with which culture plate should be recorded. 200 uL pipette tips for removing supernatent soluble protein (1 box per plate) <- make sure you have enough of these 10 mM IPTG Lysis Buffer Lysozyme DNAse and MgCl2 Plates (96 well low profile storage plate) (make sure to have plenty or extra of this type of plate since they hold plenty of volume, are still reasonably small, and don't break in the centrifuge) (note that these don't break as easily during centrifugation) (2X this type of plate for overnight culture, and protein production which is the same plate used for the insoluble protein lysate at the end of the protocol) Plates (Plain 96 well round bottom assay plates) (round bottom plates can be ordered from Denville scientific (cat no P9735) (1X this type of plate for the final soluble protein lysate) Plates (384 well plates for printing) (1X this type of plate for printing)
 * 11.1 uL needed per well or 100 uL culture
 * powder last seen at -20 C on shelf of door
 * 238.3 g/mol
 * for 40 mL 10 mM IPTG
 * 10*mmol/(1*L)*1*mol/(1000*mmol)*238.3*g/(1*mol)*1000*mg/(1*g)*0.04*L = 95.52 mg IPTG in 50 mL H20
 * 100 uL needed per well
 * 10 mL solution
 * 9.85 mL 1X PBS
 * 1% triton X-100 (so 100 uL)
 * last seen on Felicia's shelf
 * 1 mM PMSF (a protease inhibitor) (so 50 uL of 0.2 M PMSF
 * 0.348g into 10mL of isopropanol
 * last seen DNase box 3rd shelf in -20 C (powder last seen in back chemical cabinet under "P")
 * PMSF stands for phenylmethanesulfonylfluoride or phenylmethylsulfonyl fluoride, and it is a serine protease inhibitor
 * 1 complete protease inhibitor tablet (add tablet just before use)
 * last seen at 4 C fridge by door on JC's shelf
 * official name: Complete Protease Inhibitor Cocktail from Roche
 * 2.5 uL per 100 uL original culture
 * 10 mg lysozyme in 5 mL PBS for multichannel (or 60 mg for 30 mL)
 * last seen 4 C fridge
 * final concentration (10*"mg"/(5000*"uL")*2.5*"uL")/(100*"uL")*1000*"ug"/(1*"mg") = 0.05 ug/uL
 * located in 4 C
 * DNAse
 * 200 ug/mL prepared with 1 mg in 5 mL RNAse free H20
 * last seen -20 C in 15 mL tube
 * 2.5 ug/mL prepared with 375 uL of 200 ug/mL in 30 mL H20
 * powder last seen -20 C Q freezer in door in small bottle
 * MgCl2
 * 1 M liquid solution last seen on Felicia's shelf
 * comes as MgCl2*6H20 which is 203.3 g/mol (powder last seen on chemical shelf in back)
 * Example calculation: 10*mmol/(1*L)*1*L/(1000*mL)*15*mL*1*mol/(1000*mmol)*203.3*g/(1*mol) = 0.030 g
 * last seen -20 in 15 mL tube by DNAse)
 * DNAse and MgCl2 mixed solution
 * 0.2 ug/mL DNAse and 11.25 mM MgCl2
 * MgCl2
 * y*10/112.5 = 1 mM -> y = 11.25 mM
 * 1000*mM*y/30*mL = 11.25 mM -> y = 0.338 mL or 338 uL
 * DNAse
 * 200*ug/mL*y/(30000*uL) = 0.2*ug/mL -> y = 30 uL
 * 10 uL added to each well
 * final conc. MgCl2: 10*"uL"*11.25*"mM"/(100*"uL")= 1.1 mM
 * final conc. DNAse: 10*"uL"*0.2*"ug"/"mL"/(100*"uL")*1*"mL"/(1000*"ug")= 0.02 ug/mL
 * prepared by adding 0.6 uL of 200 ug/mL DNAse to 30 mL H20 and 338 uL 1 M MgCl2 to the same 30 mL.

Airpore tapesheets 3M tapesheets Polystyrene reagent reservoir LB/carb EDTA Biomek Tips Glycerol
 * 1 set used for overnight culture, 1 set used for culture to 0.5 OD, 1 set arfter IPTG has been added
 * 1 set for glycerol stock, 1 set when plates are frozen after lysozyme has been added
 * about 300 uL total need per well for whole procedure
 * about 0.25 uL of 500 mM EDTA needed per well. Final conc: 1 mM
 * last seen on chemical shelf by weights
 * P250 (Cat No 717252)
 * 4 per batch and 2 per plate
 * 1/10 dilution and glycerol stock (per plate and 2 per batch)
 * IPTG plates (per batch)
 * transfer to deep well plates and add lysis buffer (per plate)
 * add DNAse/MgCl2 (per batch)
 * P50 (A21586)
 * 2 per batch
 * add lysozyme (per batch)
 * add EDTA (per batch)

Protocols
 * Place 200 uL LB/carb with bacteria into wells of 1.2 mL deep well square storage culture plate
 * Grow overnight 37 C 250 rpm. If many plates are being handled, it may be good idea to perform this incubation as well as other incubations with the HiGro. May want to use 200 rpm instead of 250 rpm with the HiGro. If the HiGro is used, make sure to clean afterward by spraying with 70% ethanol and drying.
 * Start warming some LB/carb to use for dilution the next day.
 * Make sure to mark plates so that it is clear which wells contain which samples if any.
 * Perform dilution of culture into new plate
 * 1/100 dilution may be best. This is the dilution they do in the Molecular Cloning book protocol.
 * took about 110 min to reach OD between 0.5-1.0 with this dilution last time. 1/10 dilutions have taken about 30 min in the past.
 * a 1/100 dilution can be made by diluting 10 uL culture into 1000 uL LB
 * Make glycerol stock (may not be necessary for some experiments) (25% glycerol stock is probably appropriate)
 * Ex calculation for adding glycerol to all 200 uL: 40%*y/(200*uL+y)=25% -> y = 333.3 uL so add 333.3 uL 40% glycerol to 200 uL culture
 * Grow at 37 C 250 rpm until OD600 is about 0.5 (takes about 30 min or 2 hr depending on dilution)
 * Start thawing IPTG. Note: Don't thaw IPTG solution from -20 C to 37 C in a glass container since the glass will probably break.
 * Use multichannel pipette and polystyrene reagent reservoir to add IPTG. Alternatively, plate manipulations can be performed with the Biomek.
 * It may be best to try a range of IPTG concentrations 0.01-5 mM.
 * 11.1 uL (per 100 uL) of 10 mM IPTG for a final concentration of 1 mM IPTG.
 * Make sure to return IPTG to -20 C
 * Grow at 250 rpm or 200 rpm in the HiGro
 * It may be good to try a range of times (1, 2, 4, 6 hrs). 4 hr seems to work well for many situations
 * It may be good to try a range of temperatures (15-42 C). 37 C seems to work well for many situations.
 * Note that different media can be tried as well and that pH can also affect expression (Molecular Cloning book section 15.19)
 * Prepare lysis buffer and lysozyme solutions
 * Before harvesting some culture by centrifuging, it may be a good idea to measure the OD
 * Centrifuge at about 4063 rcf (highest speed) 10 min. Note that the regular 96 well ELISA assay plates tend to break at this centrifuge speed. Therefore, the sample should be transferred to 96 well PCR plates with pointed wells (it may be harder to see and remove the supernatent from the pellet), or into the 2 mL volume 96 well Plates for growing cultures, or the 96 well low profile storage plate.
 * Discard supernatent
 * Resuspend in lysis buffer (100 uL)
 * Add lysozyme (2.5 uL per 100 uL of original culture)
 * Be careful not to add too much lysozyme. See Notes about too much lysozyme during plate production
 * Incubate at room temperature 15 min with occasional shaking
 * Freeze -80 C 10-30 min or even longer
 * Seems to take about 3-4 hr for a whole batch of plates to thaw, and about 1 hr 30 min for a whole batch to freeze.
 * Thaw completely. If the plate will be left out for a long time, it may be a good idea to thaw at 4 C so that the protein is not sitting around too long at room temperature
 * Repeat freeze/thaw 2 more times (to help break up cell walls
 * To keep track of freeze thaw cycles: F T F T F T
 * Add DNAse/MgCl2 solution (10 uL)
 * see Materials section above for preparation of solution
 * It may be best to adjust the depth of the Biomek addition so that the liquid from the tips just barely touches the liquid in the wells.
 * Shake 4 C 1 hr. This can be done with a thermomixer, rocker, or rotator in 4 C room. Plates may need to be taped together.
 * Centrifuge at about 4063 rcf (highest speed) 20 min
 * Transfer clear supernatent to new plate.
 * a 384 well plate may be necessary for slide printing; need at least 20-30 uL volume
 * may want to add EDTA to prevent bacterial growth. Final concentration (1 mM).
 * Make a 1/10 dilution of 500 mM EDTA for 50 mM
 * 50 mM*y/(120+y) = 1 mM -> y=2.4 uL of 50 mM EDTA to 120 uL for 1 mM
 * may want to add glycerol to slow down denaturation during freezing and thawing process in the future.
 * example glycerol addition: 40%*y/(120*uL) = 2% -> y = 6 uL of 40% glycerol to about 120 uL
 * example glycerol addition 2: 3 uL of 15.3% glycerol to 20 uL for a final of 2% glycerol (15.3*3/(23) = 2)
 * Note that for checking for presence of protein in a Commassie, silver stain, or Western, it may be a good idea to load "0.15 OD units" measured from harvesting or 40 ug of suspension into gel
 * If you wish to denature the proteins, add SDS (final concentration of 1%) and heat at 60 C 1 hr.
 * Store plate at -20 C or -80 C for long term storage. Plates may be stored at 4 C around printing time (try not to let it sit at 4 C for longer than 1 week). When storing plates at -20 C or -80 C it is necessary to use aluminum foil plate sealers because the clear plate sealers just come off. Keep in mind that the more times the plates undergo freezing and thawing the more that proteins may unfold and aggregate.
 * When replacing the plate sealer lids, there may be condensation on the lid. Centrifuging the plates at 3000 rpm for 5 min will remove this condensation and prevent loss of sample.

Note: A diagram can be found on 6-20-11 of Notebook "C:\kurt\storage\CIM Research Folder\DR\2013\2-19-13\protein production diagram\scan of protein production diagram from 6-20-11 of notebook.pdf"