Hi Sweta,

A few considerations:

1. First choose the area of the gene, which you want to detect that can act as a probe.

2. BLAST it with you working organism and see if it is unique.

3. Next comes, the way you want to do it. Radioactive or Non-radioactive way ? For radioactive method choose to modify your probes with Radiolabelled Phosphates.

For non-radioactive method, you can use Digoygenin (DIG) labeling, works good for me. A DIG -PCR labeling kit from Roche is available or you can directly order modified probes.

4. Make you probe smaller, even a probe upto 700 bp works for me but smaller the better.

5. If using non-radioactive method, choose your detection system and antibody-conjugates. I use Anti-DIG Fab fragments with CSPD as a detection substrate (All ready to use available from Roche).

6. Use Nylon membrane for best results.

For further considerations, go to

https://cssportal.roche.com/LFR_PublicDocs/ras/11585614910_en_12.pdf

Cheers,

Sudip

1. Make sure your probe anneals to the DNA at the temperature you carry out the southern blot. So the Tm of the probe should be at least a few degrees higher than the annealing temperature.

2. Do a BLAST against the your test organism's genome to make sure the probe does not anneal elsewhere.

All the Best!

Thanks a lot for your reply.

Also make sure your probes are small. 200-300 bps work best for me.

Hi Sweta,

A few considerations:

1. First choose the area of the gene, which you want to detect that can act as a probe.

2. BLAST it with you working organism and see if it is unique.

3. Next comes, the way you want to do it. Radioactive or Non-radioactive way ? For radioactive method choose to modify your probes with Radiolabelled Phosphates.

For non-radioactive method, you can use Digoygenin (DIG) labeling, works good for me. A DIG -PCR labeling kit from Roche is available or you can directly order modified probes.

4. Make you probe smaller, even a probe upto 700 bp works for me but smaller the better.

5. If using non-radioactive method, choose your detection system and antibody-conjugates. I use Anti-DIG Fab fragments with CSPD as a detection substrate (All ready to use available from Roche).

6. Use Nylon membrane for best results.

For further considerations, go to

https://cssportal.roche.com/LFR_PublicDocs/ras/11585614910_en_12.pdf

Cheers,

Sudip

Hey Sudip,

Thanks for your reply. I am actually planning to use the DIG labeling kit from Roche. But I just needed help with probe designing. I hope your suggestions are helpful.

Cheers,

Sweta

You are welcome Sweta and regarding probe design, as I said you can use anything over 100 bp -1000 bp to be the best probe size. And use a labeling kit for generating it.

Cheers,

Sudip

hi all

i knew the length of  fragment  after introducing to some unique restriction enzyme, now i want to choice or design probe for these fragment. may you introduce  some website for designing probe ? 

Hi,

I used the DIG labeling kit from Roche to sinthetize my probe, is there any software to calculate/estimate the Temperatura I should use in the oven during the annealing of the probe with the digested DNA in the membrane?

My probe is 800 pb in lenght.

Afterwards I used Anti-DIG Fab fragments with CSPD as a detection substrate.

Thanks in advance! Hope s.one could help me!