Library screen
Library screen
Day 1
* Start overnight of LE392 in LB with 10mM MgSO4
Day 2
* Make 12 plates for lifts using 10,000 plaques per plate dilution
* Combine 10 l of dilution to be plated with 100 l of LE392 in 1.7 ml bullet
* Warm (no shaking) at 37°C for 15 min
* Add phage/LE392 to 3 ml lambda top agarose cooled to 50°C
* Working quickly, vortex for 10 seconds and pour all 3 ml onto a pre-warmed (37°C) NZY or LBM plate; rock plate to spread agarose out over entire surface
* Once plates are solidified, incubate plates at 37°C for 6 hours
* Prepare probe following probe labeling protocol
* After 6 hours, place plates in single layer for 1 hour at 4°C to chill
* While chilling, label nylon circles with pencil or sharpie
* Prepare denat. and neutral. filter paper/saran wrap stations
* Have small amount of 2xSSPE in square glass dish for rinsing
* Have filter paper for drying circles and filter paper size of crosslinker to put circles on (re-use both of these)
* After 1 hour, do lifts with writing side up marking membranes with 18G needle (can re-use also) (!!!! SAVE PLATES!!!!)
* Denature for 5 min from start of the first circle (writing side up)
* Neutralize for 5 min from start of the first circle (writing side up)
* Rinse in 2x SSPE and lay plaque side up on extra filter paper (writing side down)
* Let dry until no pooled liquid is visible on surface (do not overdry)
* Crosslink on auto setting in crosslinker
* Transfer circles to seal a meal bag with circles back to back (plaque side out)
* Add 15 ml hybridization solution warmed from 37°C to 42°C
* Add 200 l (133 g/ml) of 10 mg/ml sonicated salmon sperm DNA which has been heated for 5 min at 95°C
* Don't seal, just lay in hybrid oven at 42°C for at least 5 min
* Add amount of probe determined, mix, and seal bag
* Transfer bag(s) to zip lock bag
* Put in hybrid. oven and rock o/n at 42°C noting the time started
Day 3
* Note time hybridization stopped
* Cut bag and pour solution into liquid wastes using kimwipe to wipe edge
* Remove circles to small amount of 0.2x SSPE/0.1% SDS (wash sol'n) in small glass dish
* Hand agitate and pour sol'n into liquid waste, wipe edge with kimwipe
* Add 300-400 ml wash sol'n
* Agitate 20 min at 50°C in water filled hot shaker
* Pour sol'n into liquid waste, wipe edge with kimwipe
* Wash two more times (3 x 20 min washes total)
* Pour #2 and #3 wash sol'n into sink rinsing corner with hot water
* Transfer circles to filter paper
* Stretch and tape saran wrap over film cassette template
* Lay filters on wrap with writing up
* Stretch and seal wrap, trim edges
* Put in cassette with plaques down and intensifying screen below
* Put orientation labels on saran wrap to align circles with film
* Put film(notch in upper right corner if notched) between screen and circles
* Expose 4-24 hour at -70°C depending on counter detected signal
After Film is Developed
* Align labels between film and circles
* Mark needle holes with fat sharpie
* Trace over circle label, circle possible positives, and label film with date, time exposed and probe used
* For positives, align appropriate plate on film using needle marks
* Check to see if positive on film lines up with plaque on plate
* If so, pull plug of plaque and put in 1 ml of SM in 1.7 ml bullet with 1 drop of chloroform, store at 4°C
* Rescreen
Day 1
* Start overnight of LE392 in LB with 10mM MgSO4
Day 2
* Make 12 plates for lifts using 10,000 plaques per plate dilution
* Combine 10 l of dilution to be plated with 100 l of LE392 in 1.7 ml bullet
* Warm (no shaking) at 37°C for 15 min
* Add phage/LE392 to 3 ml lambda top agarose cooled to 50°C
* Working quickly, vortex for 10 seconds and pour all 3 ml onto a pre-warmed (37°C) NZY or LBM plate; rock plate to spread agarose out over entire surface
* Once plates are solidified, incubate plates at 37°C for 6 hours
* Prepare probe following probe labeling protocol
* After 6 hours, place plates in single layer for 1 hour at 4°C to chill
* While chilling, label nylon circles with pencil or sharpie
* Prepare denat. and neutral. filter paper/saran wrap stations
* Have small amount of 2xSSPE in square glass dish for rinsing
* Have filter paper for drying circles and filter paper size of crosslinker to put circles on (re-use both of these)
* After 1 hour, do lifts with writing side up marking membranes with 18G needle (can re-use also) (!!!! SAVE PLATES!!!!)
* Denature for 5 min from start of the first circle (writing side up)
* Neutralize for 5 min from start of the first circle (writing side up)
* Rinse in 2x SSPE and lay plaque side up on extra filter paper (writing side down)
* Let dry until no pooled liquid is visible on surface (do not overdry)
* Crosslink on auto setting in crosslinker
* Transfer circles to seal a meal bag with circles back to back (plaque side out)
* Add 15 ml hybridization solution warmed from 37°C to 42°C
* Add 200 l (133 g/ml) of 10 mg/ml sonicated salmon sperm DNA which has been heated for 5 min at 95°C
* Don't seal, just lay in hybrid oven at 42°C for at least 5 min
* Add amount of probe determined, mix, and seal bag
* Transfer bag(s) to zip lock bag
* Put in hybrid. oven and rock o/n at 42°C noting the time started
Day 3
* Note time hybridization stopped
* Cut bag and pour solution into liquid wastes using kimwipe to wipe edge
* Remove circles to small amount of 0.2x SSPE/0.1% SDS (wash sol'n) in small glass dish
* Hand agitate and pour sol'n into liquid waste, wipe edge with kimwipe
* Add 300-400 ml wash sol'n
* Agitate 20 min at 50°C in water filled hot shaker
* Pour sol'n into liquid waste, wipe edge with kimwipe
* Wash two more times (3 x 20 min washes total)
* Pour #2 and #3 wash sol'n into sink rinsing corner with hot water
* Transfer circles to filter paper
* Stretch and tape saran wrap over film cassette template
* Lay filters on wrap with writing up
* Stretch and seal wrap, trim edges
* Put in cassette with plaques down and intensifying screen below
* Put orientation labels on saran wrap to align circles with film
* Put film(notch in upper right corner if notched) between screen and circles
* Expose 4-24 hour at -70°C depending on counter detected signal
After Film is Developed
* Align labels between film and circles
* Mark needle holes with fat sharpie
* Trace over circle label, circle possible positives, and label film with date, time exposed and probe used
* For positives, align appropriate plate on film using needle marks
* Check to see if positive on film lines up with plaque on plate
* If so, pull plug of plaque and put in 1 ml of SM in 1.7 ml bullet with 1 drop of chloroform, store at 4°C
* Rescreen
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