Filename: LIBPHAGE.doc Written by:M. Schiller (8/5/97)
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Protocol: Phage DNA library Screening
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Preparation Of Phage Transformable Cells
Streak out a colony of XL1-blue-mrf' on a LB plate spread with 25 µl of 15 mg/ml (in
ethanol) tetracycline.
Grow over night at 37 oC and store at 4 oC for upto 2 weeks. All steps are done with
sterile technique.
Inoculate 5 ml of LB containing 10 mM MgSO4 and 0.2% maltose and grow overnight at
30 oC to ensure that the cells will not overgrow. Do not add tetracycline, it is not
compatible with Mg2+.
In the mourning grow cells at 37 oC until an OD of 0.75-1.0 is reached.
Spin the cells at 3000 x g in a table top centrifuge for 10 min and discard the supernatant.
Gently resuspend the pellet in 5 ml of 10 mM MgSO4 and measure the OD at 600 nm.
Calculate the dilution required to reach an OD of 0.5 and add that much 10 mM MgSO4.
Store on ice or at 4 oC. The cells should be fresh for titering and plating primary libraries.
Cells upto 1 week old can be used for plating 2 and 3 screens.
Titering Library
Prepare cells as above. All steps are done in a sterile manner.
Make serial dilution’s of library (10µl + 90 µl PSM) in psm buffer up to 1010.
In 5 ml glass tubes add 10 µl of diluted phage and 200 µl of OD600 = 0.5 cells, mix
gently, and incubate at 37 oC for 15 min.
Add 3.0 ml of fresh 48 oC top agar avoiding the introduction of air bubbles and pour onto
prewarmed 100 mm LB plates. Rapidly move the liquid around plate to cover the entire
surface. Let the plate harder at RT for 5 min. Incubate plate inverted at 37 oC overnight.
Count the number of plaques on each plate and considering the use of 10 µl of phage
stock calculate the titer for each plate. Average several plates to obtain an accurate titer.
Plating One Library
Prepare 20 by 150 mm plates poured with LB and allow to dry at RT for at least 2 days.
Prewarm the plates overnight and start 3 -5 ml overnight XL1blue mrf’ cultures as above.
Phage should be plated early in the morning and allowed to grow all day until desired
plaque size is reached.
Prepare cells as above. All steps are done in a sterile manner
Dilute the library in PSM buffer such that 50 µl will contain 50,000 phage based on the
previously calculated titer.
In round bottom 10 ml plastic tubes add 50 µl of diluted phage add 600 µl of OD600 = 0.5
cells, mix gently and incubate at 37 oC for 15 min.
Add 7.0 ml of fresh 48 oC top agar avoiding the introduction of air bubbles and pour onto
prewarmed 150 mm LB plates. Rapidly move the liquid around plate to cover the entire
surface. Let the plate harder at RT for 5 min. Incubate plate inverted at 37 oC until
plaques reach a diameter of 1-2 mm.
Immediately put plates in refrigerator for at least 2 hours until colony lifts are performed.
Eipper/Mains Protocol Manual
Filename: LIBPHAGE.doc Written by:M. Schiller (8/5/97)
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One And Two Screen
Perform colony lifts as described in lift protocol).
The filters should be baked rather than crosslinked with UV light.
Hybridize filters with desired probe (see DNAprobe.doc and hybe.doc).
Expose for 6 hrs - 2 days depending on the intensity of the signal determined with a
Geiger counter.
Core positive plaque from agar plate using the large end of a Pasteur pipette and transfer
it to a sterile microfuge tube containing 1000 µl of PSM buffer.
Add 20 µl of chloroform. Vortex briefly to remove phage particles and let sit at least 2
hours at room temperature. Phage are good for 1 year stored at 4 oC.
Dilute phage stock from each primary plug 1:1000 in PSM buffer.
In a glass tube, add 200 l of 0.5 OD@600/ml XL1-blue-mrf' cells and 10 µl of a 1:1000
diluted phage stock. Incubate mixture at 37 oC for 15 min.
Add 3.0 ml of fresh 48 oC top agar avoiding the introduction of air bubbles and pour onto
prewarmed 100 mm LB plates. Rapidly move the liquid around plate to cover the entire
surface. Let the plate harder at RT for 5 min. Incubate plate inverted at 37 oC overnight.
Repeat 1-6 for tertiary screen and quaternary screen if required. Screen until a phage on a
plate are positive and then excise the phage if in a lambda ZAP vector as below.
In Vivo Excision
Core positive plaque from agar plate using the large end of a pastuer pipette and transfer
it to a sterile microfuge tube containing 500 µl of PSM buffer.
Add 20 µl of chloroform. Vortex prefly to remove phage particles and let sit at least 2 h
at room temperature. Phage are good for 1 year stored at 4 oC.
In a 50 ml conical tube add 200 l of 1.0 OD@600/ml XL1-blue-mrf' cell, 200 µl of
eluted phage stock (>1x105pfu/ml, and 1 µl of Exassist helper phage (1x106pfu/ml).
Incubate mixture at 37 oC for 15 min.
Add 3 ml of 2X YT media and incubate 2 to 2.5 h at 37 oC with vigorous shaking.
(cloudy growth may not be seen)
Heat tube at 70 oC for 20 min, then spin in table top centrifuge for 15 min at 4,000 rpm.
Decant the supernatant into a sterile tube. This stock contains the plasmid packaged as a
filamentous phage particle and can be stored at 4 oC for 1-2 months.
Plate the rescued phage by adding 200 µl of SOLR cells (1.0 OD@600/ml) to two 1.5 ml
tubes.
Add 1 or 50 µl of the phage to each of the tubes and incubate at 37 oC for 15 min.
Plat 100 µl from each tube onto LB/ABP plate and incubate at 37 oC overnight.
Eipper/Mains Protocol Manual
Filename: LIBPHAGE.doc Written by:M. Schiller (8/5/97)
Chemical
maltose
MgSO4-7H2O
Vendor
Catalog #
Solutions:
PSM buffer
5.8 g NaCl, 2.0 g MgSO4-7H2O, 50 ml 1 M Tris-HCl, pH 7.5, 5 ml 2.0% gelatin. autoclave
1.0 M MgSO4
XXXX g MgSO4-7H2O + 500 ml H2O, autoclave
20% maltose
20 g maltose to 100 ml with water, autoclave
Top agar
10 g bactotryptone, 5 g bacto yeast extract, 10 g NaCl, 7 g bacto agar. autoclave.
Special notes, common problems, and troubleshooting:
References:
Eipper/Mains Protocol Manual
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