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1 Pour appropriate number of sterile LB agar-ampicillin plates on level surface
Nunc TC Dishes(245x245x25mm) will hold:-
4 separate transfer membranes per plate
Each membrane printed with colonies from up to 4 individual
microtitre plates
Print one full set of membranes for each series of probes used for
screening
It is advisable to make a copy set of membranes
2 Allow plates to set, cool and dry off
Preparation of Colony Printing Rig
1 Surface sterilise rig with 70% ethanol
2 Surface sterilise membrane plate holder with 70% ethanol
Preparation of Colony Printing hedgehog
1 Fill microtitre plate with 70% ethanol and stand hegdehog in
plate
Colony Printing-preparation of nylon transfer
membranes
Use flat-ended forceps carefully to handle nylon transfer
membranes and wear powder-free gloves, especially if using
non-radioactive probes.
1 Cut +ve charged nylon transfer membranes4 - 80x120mm
2 Cut off lower right hand corner of each membrane to allow
orientation
3 Label right-hand edge of membranes with pencil
4 Place membranes on cling-film sheet, sandwich with second layer and
trim to leave sealed edge
5 Sterilise membranes by exposure to bactericidal Ultra Violet
irradiation - 3 minutes each side
6 Carefully unwrap the membranes and transfer to sterile LB
agar-ampicillin plates
Colony Printing from microtitre culture plates
1 Transfer a membrane to the sterilised membrane plate holder
and load into printing jig
Orientate with reference to the clipped lower right corner
2 Flame sterilise the hedgehog and allow to cool
3 Immerse hedgehog in microtitre culture plate
Mix contents by gentle motion of the hedgehog in the wells
Lift hedgehog out of microtitre plate and align in printing jig
Lower hedgehog prongs keeping them parallel to membrane
Check all prongs incontact with membrane to ensure transfer of
culture supernatant
Return hedgehog to ethanol plate
Alignment of hedgehog in jig for multiple colony
printing
4 Repeat steps 2 and 3 for each plate
Contents from 4 plates can be transfered to one membrane
A copy of each membrane is required for each probe series used in
screening
5 Incubate LB agar-ampicillin plates at 3 degrees C overnight
Processing of Colony Membranes ready for
hybridisation
1 Transfer membranes with forceps, colony side up onto filter
paper5 saturated with 10% SDS
Leave 3 minutes (lysis)
2 Transfer membranes to filter paper saturated with 0.5M NaOH/1.5M
NaCl
Leave 5 minutes (denaturation)
3 Transfer membranes to filter paper saturated with 0.5M Tris, pH
6.8/1.5M NaCl
Leave 5 minutes (neutralisation)
4 Transfer membranes to filter paper saturated with x2 SSC6
Leave 5 minutes
5 Transfer to fresh filter paper and AIR dry
Leave ~30 minutes
6 Bake at 80 degrees C for 2hr
7 Store membranes at -20 degrees C
LB AGAR (PER LITER)
10g of NaCI
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 degrees C before addition of
antibiotic
Pour into petri dishes (~25 ml/100-mm plate)
120ml/245x245x25mm plate; holds 4 filters each (TC
DISH #66508; Nunc, Denmark)
Ampicillin at 50 microgrammes/ml
Hybond-N+ (Nylon transfer membrane; Amersham, UK)
3MM Chromatography paper (Whatman International Ltd., UK)
x20 SSC: 174g/litre NaCl
88.2g/litre Na3Citrate
adjusted to pH 7.0