4.6@ Library Screening

Yuji Hiwatashi and Keiko Sakakibara

 

Introduction

cDNA library screening allows detection of expressed genes for subsequent cloning and sequencing.@ This protocol describes screening of cDNA library lambda gt11.

 

Material required

E 14 cmx10 cm plastic plate (eikenkagaku No.2)

E 9 cm-plastic dish

E Test tube (r = 12 mm) (autoclave)

E 14 ml conical tube

E tooth tips (autoclave)

E Forceps

E Pipett (autoclave)

E 1.5 ml micro tube (autoclave)

E 100 ml flask (autoclave)

E E.coli strain Y1090

E LB@ (autoclave)

E 1 M MgSO4 (filtered with a 0.22 µm unit)

E 10 mM MgSO4 (filtered with a 0.22 µm unit)

E SM buffer

E Chloroform

E TE-saturated phenol

E Lambda plate (1 g tryptone, 0.5 g NaCl, 1.5 g agarose / 100 ml H2O)

E Top agar (1 g tryptone, 0.5 g NaCl, 0.4 g agarose / 100 ml H2O)

E Amersham Hybond-N+ nylon membrane

E Whatmann 3MM chromatography paper

E Denaturing solution (1.5 M NaClA0.5 M NaOH)

E Neutralizing solution (1.5 M NaClA0.5 M Tris-HCl (pH 8.0)A0.001 M EDTA (pH8.0))

E 20x SSC

E 10% SDS

E 0.5 M EDTA pH 8.0

E Agarose

E QIAGEN gel extraction kit

 

Procedure

1. Culture of host cells

a) Streak Y1090 from frozen stock on an LB plate.@ Incubate plate overnight at 37˚C.

b) Pick a single Y1090 colony with a sterile toothpick to inoculate 5 ml of LB containing 50 mg/l ampicilin in a sterile 15 ml test tube.@ Grow overnight at 37˚C.

c) Pour 1 ml of the culture to a 1.5 ml micro tube and place on ice for 5 min.

d) Spin at 5000 rpm for 5 min.

e) Discard the supernatant and re-suspend gently pellet in 1 ml of 10 mM MgSO4. Transfer the suspension to another tube, then re-suspend pellet to make 2x concentration (2x host cells). Store diluted cells at 4˚C (use within 2-3 days)

 

2. Infection and plating

a) To titer phage library, thaw phage. Add 3 µl of 10-fold dilution of phage (until to 1/2000; dilute in 10 mL MgSO4) to 600 µl of 2x host cells (from step 1-e) in a sterile 15 ml conical tube. Mix gently, then incubate at 37˚C for 20 min. During this waiting time, melt top agarose and have it cooled to 50˚C. Add 6 ml of 50˚C top agarose to the first tube, mix by inversion several times, then quickly pour onto the center of a 14x10 mm lamda plate. Repeat with the next tubes. After all plates have been poured and hardened, invert and incubate at 37˚C. Count number of plaques after 4-5 hrs.

b) Using same host cells from step 1-e, dilute phage to give 20,000 cfu/14x10 mm plate. Do 10 plates, as - described in 2-a. Incubate at 37˚C for 4-5 hrs.

c) Store at 4˚C for more than 2 hrs.

 

3. Plaque lift

a) Soak 2 sheets (20 x 30 cm) of Whatmann 3MM with denaturing solution and neutralizing solution, respectively.

b) Label 14 x 9 cm Hybond N+ membrane with fine tip pencil to correspond to plate. Holding membrane at edge with forceps, place membrane onto plaque surface. Mark filter in 3 asymmetric locations by stabbing through it and agar with 18G needle. After 30 sec, pull off membrane and immerse DNA side up for 30 sec onto a 3MM paper containing denaturing solution. During denaturation, place new membrane for 30 sec as described above.@

c) Remove and transfer onto a paper containing neutralizing solution for 5 min.@ Repeat this step.@

d) Rinse membrane with 2x SSC.@

 

4. Hybridization

See protocols for genomic southern analysis using RI and non-RI.@

 

5. Second screening

a) Pick positive plaques by stabling with a blue tip (for P1000), and squirt into 1 ml of SM.

b) Measure titer of the phage solution.

c) Add the diluted phage solution (give 100 plaque per plate) with 600 µl of 2x host cells Y1090 (as step 1-e), and incubate at 37˚C for 20 min. Add 3 ml of 50˚C top agarose, mix by inversion, then quickly pour onto a lambda plate (9 cm-dish). Incubate at 37˚C for 4-5 hrs, then place at 4˚C.

d) Do plaque lift and hybridization as described above.

@

6. Sub-cloning of insert from a positive phage.@

a) Pick positive plaques by stabling with a blue tip (for P1000), and squirt into 1 ml of SM.

b) Add the diluted phage solution with 2x host cells (step1-e). After incubation at 37˚C for 20 min, add the mixture into 10 ml LB supplemented with 100 mg/l ampicilin and 10 mM MgSO4 in 100 ml flask.@

c) Incubate at 37˚C. Check optical density every one hr. When the culture becomes transparent (probably it will take 4-5 hrs), add 2 ml of chloroform.

d) Transfer the culture into micro tubes and centrifuge at 10000 rpm for 5 min.@

e) Transfer the supernatant into ultracentrifuge tube and ultracentrifuge at 26000 rpm for 30 min (Hitachi, rotor no. RPS55T2).@

f) After decanting supernatant, suspend pellet in 250 µl of SM, then add 5 µl of 10% SDS and 5 µl of 0.5 M EDTA.

g) Extract it with phenol and then precipitate with ethanol. Dissolve pellet in 20 µl of water.@

h) Digest DNA with appropriate restriction enzymes (EcoRI), followed by agarose electrophoresis.@ Recover a cDNA fragment from a gel with QIAGEN gel extraction kit or equivalents. Sub-clone it with pBluescript

i) Perform sequencing and determine sequence of a positive cDNA.