Colony+Hybridization+Protocol+3-15-12

Colony Hybridization Protocol
 * Approximate timing of experiment
 * Lift colonies -> Incubate 30 min -> Add IPTG -> Incubate 3-4 hr -> Lyse bacteria (2-3 hours) -> Block (30 min or overnight) -> Primary (1.5 hr) -> Secondary (1.5 hr) -> Scan with Typhoon (30 min)
 * Materials
 * 10 mM IPTG
 * 20 mL IPTG into 180 mL Carb
 * powder last seen at -20 C on shelf of door
 * 238.3 g/mol
 * for 50 mL 10 mM IPTG
 * 10*mmol/(1*L)*1*mol/(1000*mmol)*238.3*g/(1*mol)*1000*mg/(1*g)*0.05*L = 119.2 mg IPTG in 50 mL H20
 * Lysis Buffer
 * 200 mL solution
 * 197 mL 1X PBS
 * 1% triton X-100 (so 2000 uL)
 * last seen on Felicia's shelf
 * 1 mM PMSF (a protease inhibitor) (so 1000 uL of 0.2 M PMSF
 * 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
 * 20 complete protease inhibitor tablet (add tablet just before use)
 * last seen at 4 C fridge by door on JC's shelf
 * Lysozyme
 * 10 mg lysozyme in 5 mL PBS for multichannel (or 60 mg for 30 mL)
 * last seen 4 C fridge
 * DNAse and MgCl2
 * Example preparation: Add 35.6 uL of 0.2 mg/mL DNAse and 400 uL 1 M MgCl2 to 400 mL 1X PBS
 * DNAse (final concentration desired 0.0178 ug/mL)
 * 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 (final concentration desired 1 mM)
 * 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)
 * LB/carb
 * EDTA (final concentration desired (1 mM))
 * 500mM*y/200000uL=1mM
 * last seen on chemical shelf by weights
 * LB Plates (for titrating to determine glycerol stock concentration
 * Bioassay tray (245X245 mm^2)
 * Blot paper for drying
 * nitrocellulose blotting membrane for lifts
 * Plate serial dilutions of glycerol stock onto a large 245X245 mm^2 bioassay tray (perhaps 2 10^(-2), 2 10^(-4), and 2 10^(-6) dilutions).
 * Spread about 382 uL (derived from: 50uL/(pi*50mm^2)=y/(245*245^2mm^2) -> 50uL/7845mm^2=y/60025mm^2 -> y = 382uL)
 * Approximate Concentrations of Tumor cDNA Library Glycerol Stocks from past experiments can be found at that link
 * Make sure to return glycerol stock to -20 C
 * Gently apply the nitrocellulose filter onto the plate with colonies to "lift" the colonies. Don't press too hard on the filter to avoid damaging/smearing/removing the original colonies. Puncture a pattern of holes through nitrocellulose into LB plate with colonies so that these holes can be used to align the final nitrocellulose image with the actual colonies on the plate later on to identify specific colonies (perhaps puncture a square pattern of holes on top and a triangle pattern on the right). Place plate back into 37 C so that colonies can be picked the next day.
 * Place the colonies into a container containing 180mL LB/carb
 * Incubate with slow shaking (25 rpm) at 37 C for 30 min.
 * Add IPTG to LB for a final concentration of 1 mM
 * Example Calc: (10mM*y/200*mL=1mM -> y=20mL of 10mM IPTG in 180mL LB/Carb
 * Incubate the filter for approximately 3-4 hr 37 C 25 rpm
 * Prepare lysis buffer (probably 200 mL). Note that it is probably best to use one lysis buffer solution for a whole batch of plates so that the minimum amount of protease inhibitor tablets and other reagents is used.
 * Drain away LB in container. May want to rinse container and filter several times with PBS. Add lysis buffer (probably 100 mL)
 * Add 2.5 mL of lysozyme solution
 * Incubate at room temperature 15 min with occasional shaking
 * Add DNAse/MgCl2 solution
 * Rotate gently at 4 C 1 hr. This can be done with a thermomixer, rocker, or rotator in 4 C room. Discard solution
 * May want to add EDTA to prevent bacterial growth on membrane if it is going to be stored (final concentration 1 mM)
 * Incubate RT or 4 C with gentle shaking 15 min. Discard solution.
 * Block filter with 1% BSA in TBST 30 min - 2 hr (or overnight at 4 C if desired). Use a platform shaker to gently swirl the solution. Discard BSA/TBST solution.
 * Add primary antibody diluted 1:1,000 in 5% BSA in TBST for 1-2 hr at room temp. You will probably want to save this primary antibody solution afterward.
 * Wash 3X with TBST (may want to wash on rocker for 5 min intervals) (try not to add TBST so harshly that it washes some colonies away)
 * Apply direct labeled secondary antibody (often goat anti-mouse AF647). Dilute biotinylated secondary antibody 1:10,000 in 5% BSA in TBST and incubate 1 hr RT with shaking. You will probably want to save this secondary solution afterward.
 * Wash 3X with TBST (may want to wash on rocker for 5 min intervals) (try not to add TBST so harshly that it washes some colonies away)
 * Dry the filter between a sponge filter
 * Image with Typhoon
 * Turn on the computer for the Typhoon and select the Typhoon Scanner icon.
 * Set to fluorescence
 * Click Setup
 * Deselect laser options
 * Select the appropriate Emission and excitation lasers
 * Last Settings used were: Excitation: 633 nm, Emission filter: 670 nm, Dye: AF647
 * May want to reduce the PMT to 350
 * Orient membrane on scanner
 * Select blocks to scan and click "scan"
 * Ensure to save file on server or flash drive instead of on the computer or the file will be deleted later. Note that the standard .gel files do not open well in other programs so make sure to at least save a .tif file.
 * Once colonies are selected, pick them from the plate inoculate them into 1 mL culture 37 C 250 rpm overnight (may be best to inoculate in afternoon rather than early morning).
 * Perform plasmid miniprep
 * Sequence plasmids (Sequencing Protocol)

Other documentation Initial Protocol from Mara 3-12-12