can you tell us what the tracks are please?.

It looks like 2 ladders running well and one sample giving bands at high molecular weights. Are there more samples. What type of DNA and what enzymes do you cut with. What is the stringency of your membrane washes. What is your probe

thank you

paul

tank you for your attention . we use P13E11 as a probe which dr lemmer sent us from Germany. at first we digest our sample by ECOR1 for 25 hours then we run 2 ladders (1kb, CHEF DNA size marker) and our samples for 27 hours. we use 0.7 % AGAROSE GEL ELECTROPHORESIS. then we Put the gel on the UV transilluminator. then we denature the gel 2 times with alkalisolution each time is 15 minutes.after that we put our membrane for 5 minutes in the alkali solution. we use Nylon Membranes, positively charged from Roche Life Science. then we make the southern blot bridge and let it for about 20 hours. then we put the membrane in the neutralization buffer for 5 minutes then we put it in the uv transluminator . after that we put the membrane in the hybridization granules (roche company)for 3 hours . we put the (50microliter h2o+ 1 microliter lader chef labeled (roche company)+2microliter probe which is labeled by us (658ng/microliter))in the 100 centigrade degree for 10 minutes then put it in ice bath for 5 minutes and then pour this in the new granule then we put in in the 46 centigrade degree bath for 18 hours . then we wash the membrane 2 time (each time is 10 minutes) in wash 1 (Wash buffer I (2 * SSC/0.1% SDS) for 2 litres. ) and then 2 times in wash 2(Wash buffer II (1 * SSC/0.1% SDS) for 2 litres) the temperature is 55 centigrade degree. then we put it briefly in the maleic acid. then we put it in the block buffer(roche company)for 2 hours room temorature. after that we put it in Ab+block buffer for 30 minutes. then we wash it 2 times each time 10 minutes with wash buffer (roche company) then let it be in the detection buffer (roche company )for 10 minutes. then pour CDPSTAR on the membrane for 15 minutes and we use film to detect

thank you for that detailed response Sara . You are using a probe which should anneal diagnostically on Chr4 and also on Chr10 so even if the locus is deleted on Chr 4 we should still be seeing a signal from the constant region of Chr10. Clearly transfer of DNA has been good as the ladders show and annealing of probe is good as seen on the ladders. Hopefully you have a picture from the transilluminator showing good amounts of digested DNA in you cut samples on the CHEF gel...if these tracks are very faint then you have not used enough genomic dna. I think that you will have used the right amount of dna ( >5ug) and the correct enzyme so either the probe has washed off ( try lower wash temperatures or higher salt concentrations to get a signal and then clean it up later when you have something to work on. Meanwhile design 2 pcr primers within the p13e11 locus and ensure that you can pcr the correct thing from the material sent to you. ( errors can sometimes happen and the wrong probe may have been sent or a plasmid with the probe missing or probe at the wrong concentration perhaps. As a quicker test do not bother with the CHEF just cut some dna  and dot blot it on to a membrane in alkaline solution  and fix it. Then you can anneal at different salt or temperature concentrations just to see if you can get annealing with a stronger signal all in one place while you sort out where the problem lies.

good luck,

Paul

I am thankful with your guidance.I hope that your guidance is effective.I will tell you the result

hi dear paul. I have good news for you . We were able to discover solutions after one year. I will show you the picture . please see the attached file

Hi Sara,

that is so much better. It is no fun having a technique not working for so long. Well done you now have something that you can really work with. What do you think was the main problem?

we have alot of characters . we decreased washing temprature and changed the hybridization temprature. we also changed the amount of the probe . it depends on our probe purity . we should purify the probe then use it.