This is a cached page for the URL (http://www.bs.jhmi.edu/MBG/SeydouxLab/Protocols/probe.html). To see the most recent version of this page, please click here.
Protocol Online is not affiliated with the authors of this page nor responsible for its content.
About Cache
Probes

Back to Seydoux Lab Homepage

Preparation of single-stranded probes from cloned cDNA

(from Patel and Goodman, 1992)

a) 2-5 µg of plasmid DNA containing the cDNA insert is linearized using an appropriate restriction enzyme. For antisense probes, a unique restriction site 5' to the insert is used. This digested DNA will be amplified using an antisense primer at the 3' end of the insert. For sense (control) probes, a unique restriction site 3' to the insert is used. This digested DNA will be amplified using a sense primer at the 5' end of the insert. (Inserts of up to 2kb are labeled efficiently).

b) Add 4X STOP to digested DNA to a final concentration of 1X STOP.

Extract once with phenol/chloroform, once with chloroform, and precipitate with 3 volumes of 100% EtOH.

Resuspend in TE at a final concentration of 100-200 µg/ml. (check concentration on a gel).

c) Mix the following reagents in 0.5 ml eppendorf tube.

water 7.0 µl

10x Taq Buffer 2.5 µl

25 mM MgCl2 1.5 µl

10 x DIG-labeled dNTP mix 5.0 µl

Primer * (30 ng/µl) 5.0 µl

Digested DNA (100-200 µg/ml) 2.0 µl

mineral oil 40 µl

* For cDNAs cloned in Bluscript, we use the following primers (21mers):

"T3 long " = 5'- ACT AAA GGG AAC AAA AGC TGG -3'

"T7 long " = 5'- ACT CAC TAT AGG GCG AAT TGG -3'

d. Boil reagents for 5 min, place on ice, before adding 2.0 µl of a 1:8 dilution in water of 5 units/µl Taq polymerase stock (1.25 units total).

e. Incubate labelling reaction for 35 thermal cycles as follows:

95°C for 45 seconds

55°C for 30 seconds (lower temperature for primers less than 20nt)

72°C for 1 minute

f. 75 µl of H20 is added to the reaction below the oil and 90 - 95 µl of the diluted reaction is transferred to a new tube.

g. 10 µl of 1M NaCl, 10 µg of glycogen, and 3 vols of 100% EtOH are added to the diluted reaction. After 30 min at -70°C, the reaction is centrifuged at 15,000 rpm for 10 min. The pellet is washed in 70% ethanol, dried, and resuspended in 300 µl of hybridization buffer.

h. The probe is boiled for 1-2 hours. This step reduces the length of the probe for efficient penetration of embryos.

e. Probe production is assayed using the following protocol. 1 µl of probe in hybridization buffer is mixed with 5 µl of 5 x SSC, boiled for 5 min, and cooled on ice. 1 µl of this mixture is spotted on a nitrocellulose strip. Several dilutions of a pre-labelled control DNA (1ng to 1pg / µl; Boeringher Mannheim) are also spotted for comparison. The strip is baked for 30 min in a vacuum oven at 80oC, washed once in 2 x SSC, twice in PBT, and blocked for 30 minutes in PBT. The strip is then incubated for 30-60 min with AP-anti-DIG antibody diluted 1:2000 in PBT. After three 10 min washes in PBT, and two 5 min washes in staining solution, the strip is developed in staining solution containing 4.5 µl NBT/ml, 3.5 µl X-phosphate/ml. Spots should be visible within minutes. Spot intensities of the probe and control dilutions are compared to determine the concentration of the probe.

f. Probes can be stored at -20oC in hybridization buffer for several weeks.