cDNA library
screening allows detection of expressed genes for subsequent cloning and
sequencing.@ This protocol describes
screening of cDNA library lambda gt11.
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 NaClA0.5 M NaOH)
E Neutralizing solution
(1.5 M NaClA0.5 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
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.