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From titration results for phagemid stock set up 12-15 plates
using sufficient supernatant to generate 100-200 colonies/plate
DAY 1-Prepare overnight SOLR cell cultures
Bacterial cultures should be started from a fresh colony plate
(<7days old) using a single colony.
1 Inoculate 50ml of LB broth (without supplements) with a single
colony of SOLR cells.
2 Grow overnight cultures of the SOLR cells with vigorous shaking at
30 -37 C.
The lower temperature ensures that the cells will not overgrow.
3 Prepare 12-15 LB-ampicillin agar plates
DAY 2-Generation of colonies from phagemid stock in
SOLR strain cells.
1 Gently spin down the SOLR cells (1000 x g for 10
minutes).
2 Resuspend the cells in ~15 ml of 10 mM MgSO4. Do not vortex.
3 Dilute SOLR cells to an OD600 of 1.0 in 10 mM MgSO4.
Highest efficiencies are obtained from freshly prepared cells.
4 Prewarm and dry LB-ampicillin agar plates ready for plating in step
9
5 Add 3ml of freshly grown SOLR cells from step 3 (OD600= 1.0) to
sterile Universal tube.
6 Make suitable working dilution of phagemid stock (calculated from
titration results)
7 Add appropriate volume of phagemid supernatant (calculated from
titration results)
8 Incubate the tubes at 37 C for 15 minutes.
9 Add 200 microl from each tube to replicate LB-ampicillin agar
plates
and distribute evenly over plate surface with sterile glass
spreader
Allow excess media to dry before inverting plates
10 Incubate overnight at 37 C.
DAY 3-Picking colonies to microtitre plates
1 Check colonies have grown at expected density
2 Label 20 plates in duplicate (2 x 10 plates) with unique identifier
and date
for example: MB4SLA1 ie MB(initials)4SL(library nomenclature)A1(plate
no.)
3 Add 50 microl LB-amp broth1 to one set of duplicate plates
4 Use sterile white tips to lift single colonies from LB-amp agar
plates to individual wells of a microtitre plate. Leave tip in well
to prevent multiple inoculation.
5 Remove tips from first plate when second plate completed, remove
tips from second plate when third plate completed, and so on, this
ensures efficient inoculation of the medium.
6 Incubate at 37 C. Growth is usually visible after 4-6hr.
7 Fill spare microtire plate with 70% ethanol and stand hedgehog in
ethanol
Flame sterilise the hegehog and allow to cool
Immerse hedgehog in primary culture plate
Mix contents by gentle motion of hedgehog prongs in the wells
Inoculate matched replicate plate using hedgehog to transfer culture
suspension
Return hedgehog to ethanol plate, flame sterilise and allow to
cool
Repeat inoculation protocol for each primary plate
Double check inoculation is of labelled matched replicate plate
8 Make colony lifts on to +ve charged nylon membranes
9 Add 50 microl fresh LB-amp broth1 to primary set of duplicate
plates ie total well volume 100 microl
10 Incubate all plates overnight at 37 C
DAY 4-Store bacterial culture plates and process
colony lift membranes for screening
1 Check growth pattern same in replicate plates
2 Add 50 microl 45% sterile glycerol/LB to all wells
3 Flame sterilise the hegehog and allow to cool
Mix well contents by gentle motion of the hedgehog in the well
4 Repeat step 2 for each plate
5 Store primary culture plate as Archive plate at -80 C
Store secondary plate as Working copy plate at -80 C
LB BROTH (PER LITER)
10g of NaCl
10g of bacto-tryptone
5g of bacto-yeast extract
Adjust to pH 7.0 with 5 N NaOH
Add deionized H2O to a final vol. of 1 liter
Autoclave
Allow medium to cool to 55 C before addition of antibiotic
[Ampicillin] 50µg/ml
LB AGAR (PER LITER)
10g ofNaCl
10g of bacto-tryptone
5g of bacto-yeast extract
15g of bacto-agar
Adjust pH to 7.0 with 5 N NaOH
Add deionized H2O to a final vol. of 1 liter
Autoclave
Allow medium to cool to 55 C before addition of antibiotic
Pour into petri dishes(~25 ml/100-mm plate)