cDNA
library screening allows detection of expressed genes for subsequent cloning
and sequencing.@ This protocol describes
screening of a cDNA library in lambda gt11.
E 14 cm x 10
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 picks
(autoclave)
E Forceps
E Pipette (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 ampicillin 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 pellet gently 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 serially diluted phage (lowest
dilution 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 cm lamda plate and rock gently to cover the agar surface. 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 the same
host cells from step 1-e, dilute phage to give 20,000 cfu/14x10 cm plate. Prepare
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+ membranes with a fine tip pencil to correspond to each plate.
Holding the membrane at the edge with forceps, place the membrane onto the plaque
surface. Mark the filter in 3 asymmetric locations by stabbing through it and the
agar with an 18G needle. After 30 sec, pull off the membrane and place it, DNA
side up, for 30 sec on a 3MM paper soaked with denaturing solution. During
denaturation, place new membrane on the agar surface for 30 sec as described
above.@
c) Remove the
membrane from the denaturing solution and transfer onto a paper soaked with
neutralizing solution for 5 min.@
d) Rinse
membrane with 2x SSC.@ Continue to cycle
further membranes from agar to denaturing solution to neutralising solution to
2x SSC.
4. Hybridization
See
protocols for genomic southern analysis using RI and non-RI.@
5. Second
screening
a) Pick
positive plaques by stabbing 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 (to give 100 plaques 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 stabbing 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
ampicillin and 10 mM MgSO4 in a 100 ml flask.@
c) Incubate
at 37˚C. Check optical density every hour. 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 supernatants into an ultracentrifuge tube and ultracentrifuge at 26000 rpm
for 30 min (Hitachi, rotor no. RPS55T2) to pellet phage particles.@
f) After
decanting supernatant, suspend the pellet in 250 µl of SM, then add 5
µl of 10% SDS and 5 µl of 0.5 M EDTA.
g) Extract phage
DNA with phenol and then precipitate with ethanol. Dissolve pellet in 20
µl of water.@
h) Digest the
DNA with appropriate restriction enzymes (EcoRI cuts at the cloning site),
followed by agarose electrophoresis.@
Recover the cDNA fragment from the gel with a QIAGEN gel extraction kit
or equivalent. Sub-clone it with pBluescript
i) Perform
sequencing and determine sequence of a positive cDNA.