Protocol L.1

Lambda gt22a Screening

This protocol is a modified version of the one described by Vinson et al. (G&D; 2:801-806, 1988). Specifically, I have modified the probe preparation and the buffer system.

 

Solutions

 

T-TYN Media + Mg+2

10 g tryptone

5 g yeast extract

5 g NaCl

10 ml 1M Tris pH 7.2

bring up to 1 liter with water, adjust the pH to 7.2 with NaOH,

autoclave, cool and add 10 ml 1M MgCl2

NOTE: FOR GROWING UP CULTURES OF Y1090r- ADD 1 ml 20% MALTOSE PER 100ml

 

T-TYN Plates

1 liter T-TYN Media

add 15 g agar

autoclave, cool and add 10 ml 1M MgCl2

pour 10 large plates

 

T-TYN Top Agarose

make 1 liter T-TYN Media

add 7 g Ultrapure Agarose

autoclave, cool and add 10 ml 1M MgCl2

use 9-10 ml per large plate

 

 

Phage Dilution Buffer (PDB)

50 mM Tris 7.5 5.0 ml 1M Tris pH 7.5

100 mM NaCl 2.0 ml 5M NaCl

10 mM MgCl2 1.0 ml 1M MgCl2

0.01% gelatin 0.01 g gelatin

up to 100 ml with Q

 

 

10X Kinase Buffer (-DTT)

700 mM Tris 7.5 700 ml 1M Tris 7.5

100 mM MgCl2 100 ml 1M MgCl2

200 ml Q

 

Calf Thymus DNA

sonicate the entire bottle in 25 ml Q

pass through a 25ga. needle until homogenized

phenol/chloroform extract

EtOH precipitate with 0.1 vol. 1M NaCl and 2 vol. EtOH

wash and dry

resuspend to approx. 10 mg/ml and quantitate by OD260

NOTE: BEFORE USE, BOIL FOR 5' AND HOLD ON ICE

 

 

 

10X Binding Buffer

100 mM Tris 7.5 400 ml 1M Tris 7.5

500 mM KCl 150 g KCl

up to 4 liters with Q

Denaturing Solution

1146 g guanidine HCl (From Aldrich Chemicals)

200 ml 10X Binding Buffer

2 ml 0.5 M DTT

up to 2 liters with Q

 

 

 

1X Binding Buffer

100 ml 10X Binding Buffer

900 ml Q

2 ml 0.5 M DTT

 

 

Prehybridization Solution

200 ml 10X Binding Buffer

100 g nonfat dry milk

2 ml 0.5 M DTT

1800 ml Q

stir for 0.5-1.0 hour to dissolve milk

 

 

Wash Solution

200 ml 10X Binding Buffer

5 g nonfat dry milk

2 ml 0.5 M DTT

1800 ml Q

Stir for 0.5-1.0 hour to dissolve milk

(For hybridization, add probe and boiled calf thymus DNA(25 mg/ml final concentration))

 

 

Procedure

 

 

plate phage

Plug an overnight culture of Y1090r- in T-TYN (Maltose,Mg+2,Amp). Dilute the overnight culture approximately 2-fold, check the OD600, and adjust as needed to an OD600 of 0.5. Mix 450 ml of this culture with 20,000 pfu of the library and incubate at 37° for 20-30 minutes. Add 8-9 ml of T-TYN Top Agarose and pour onto prewarmed 150 mm plates. Incubate at 43° for 6 hours. It is best to start this at midnight and let it go until early morning.

• Soak the Nitrocellulose Filters (S&S NC45) in 10mM IPTG (0.238 g/100 ml) for 5 minutes. If it is more convenient these can be stored overnight in the IPTG at 4°. Dry the filters for 5-10 minutes and overlay onto the plates. Immediately mark the plates with an 18ga. needle and mark the holes on the bottom of the plate with a pen. Incubate these plates for 4 hours at 37°.

• Remove the filters and set them out to dry until the duplicates are ready. Overlay the duplicate filters (also soaked in 10mM IPTG) and mark in the same spot with the 18ga. needle. Allow these to incubate for 1-2 hours. Remove the filters and set them out to dry for approx. 15 minutes. (All of the filters should be ready by about 1:00

 

 

probe preparation

• The probe should be complementary oligonucleotides corresponding to the protein binding site with BamHI and BglII half sites. Gel purify the oligos and kinase each one by mixing the following:

5 mg oligo

5 ml 10X Kinase Buffer

2 ml Polynucleotide Kinase

2.5 ml 0.1 M DTT

1 ml 10mM ATP

Q to 50 ml

Incubate for 30 minutes at 37°. Mix the complementary oligos and heat to 65° for 2 minutes. Slow cool the tube in 50 ml 65° water for 30 minutes. Phenol/chloroform extract and EtOH (no carrier) precipitate the annealed oligos (10 mg total ds DNA).

• Ligate the oligonucleotides overnight at 16°, followed by an extraction and EtOH precipitation. Digest the Multimers with BamHI and BglII for 4 hours and run 0.5 mg on a 10% acrylamide gel to check efficiency of ligation and cutting.

• Boil 1-2 mg probe in 11 ul Q for 5’ and hold on ice. Add 24 ml A:B:C, 4 ml BSA, 5 ml each a32PdATP and a32PdCTP and 1 ml Klenow. Incubate at room temperature for 1 hour. Add 170 ml TE, 8 ml EDTA, and 1 ml tRNA. Phenol/chloroform extract and EtOH ppt with 0.5 volume 7.5 M NH4OAc. Repeat the precipitation 1-2 times to remove the free nucleotides or use a purification column. For more details see the random prime protocol.

 

denature proteins

NOTE: Not all proteins bind better when subjected to renaturation so the appropriated controls should be performed.

• Add filters one at a time (no more than 20 per dish) to the appropriate volume of Denaturing Solution:

500 ml in 190x100 crystallization dish

100 ml in 125x65 crystallization dish

Shake for 5 minutes at room temperature at about 45 rpm, and repeat.

• Dilute the denaturing solution two-fold with 1X Binding Buffer and add the filters one at a time. Shake for 5 minutes at room temperature at about 45 rpm, repeat 5 times.

• Wash with 1 liter or 500 ml of 1X Binding Buffer for 5 minutes at room temperature at about 45 rpm, repeat.

 

prehybridize

• Add 1 liter or 500 ml of Prehybridization Solution and shake overnight at 4°.

• Wash with 1 liter or 500 ml wash solution at room temperature for 5 minutes.

 

hybridize

• Add the labeled probe (0.5 mg/100 ml is ideal, see R.1) and 25 mg/ml boiled Calf Thymus DNA to the appropriate volume of Wash Buffer. Shake for 2 hours at room temperature, and wash with 2-4 liters of Wash Buffer.

• Dry the filters, mount them on bleached autorads, and expose for 48 hours with a screen.