Your probe is too short to be labelled using nick translation. It is much better to label the probe by PCR. With nick translation, if well done, you should see a smear of DNA fragments on the gel, in your case a smear of very short fragments.

thanks Marec sir

Can you please suggest me the protocol for labelling by PCR. Using nick translation i had obtained results like complete smearing or a band of

Dear Sulochana,

we use the following mixtures for PCR labelling of gene fragments (cca 500-1000 bp long) with fluorochrome-dUTP:

Stock dNTP mix:

100mM dATP 3.5 μl

100mM dGTP 3.5 μl

100mM dCTP 3.5 μl

100mM dTTP 1.0 μl

water 338.5 μl

total 350 μl

50 μl PCR mix:

template DNA 1 μl

primer F (100 μM) 0,5 μl

primer R (100 μM) 0,5 μl

ExTaq buffer 5 μl

stock dNTP mix 5 μl

fluorochrome-dUTP 1 μl

ExTaq polymerase 0,5 μl

water 36.5 μl

PCR labelling using this PCR mix can be done under the same PCR conditions that you use for amplification of the gene fragment.

Hope it helps.

Best regards,

Frantisek

Dear Frantisek sir,

Thanks for valuable suggestion. Since the material i am provided with is not exactly the same as mentioned by you in protocol. Can components such as dNTP mix can be replaced by Master mix (Fermentas) containg dNTP's, Taq pol and buffer. Since i do not have fluorochrome-dUTP, i have only flourescent dye such as Alexa Flour is it possible to substitute for fluorochrome-dUTP.

secondly, what is meant by cca 500-1000 bp long

Dear Sulochana,

(1) No, you cannot replace dNTP mix for probe labelling with dNTP's from Fermentas Master mix. The problem is that in the Master mix there is a 1:1:1:1 ratio between nucleotides, and for labelling you need a mix with a lower concentration of dTTP, which will allow to incorporate fluorochrome dUTP into the amplified DNA product.

(2) No, it is impossible to replace flourochrome-dUPT with flourochrome only. Such fluorochrome (for example Alexa) woud stain DNA but not label the DNA.

(3) 500-1000 bp long means the size of used DNA fragments.

Hope it helps.

Best regards,

Frantisek

Dear Frantisek sir,

Thanks a lot sir for co-operating in my experiment, its because of your valuable suggestion i will proceed in a new direction and hope optimistically in this direction.

There are few more quries like, will the rest of the procedure for hybridisation will be simoliar to usually followed produre in FISH experiments.

secondly, dye such as DAPI (358⁄461) can be used as counterstain along with fluorescein-12-dUTP (495/525 nm) in staining

Dear Sulochana,

You may use a general FISH protocol with some modofications that depend on the probe, type of probe labelling (but not on the labelling method), and target sequences to be localized on chromosomes. With "type of labelling" I mean whether the probe is labelled with fluorochrome (which is your case) or with a small "non-fluorescent" molecule such as biotin or digoxigenin (the latter type has to be detected using fluorochrome-conjugated antibodies.

DAPI is fine for counterstaining.

Good luck,

Frantisek