Dear Andrea,

Thank you!

To begin with, I use Alkaline lysis BAC prep based on protocol I adopted from National Institute of Mental Health, Neuroscience website, which gives me high quality and quantity DNA and therefore quantity is generally not an issue for me. However, quality sometimes has been an issue.

I use about 1-2 ug of DNA for 20 ul nick translation reaction.

I use dUTP-dig or dUTP-biotin for labeling of the DNA. I don't use DTT, and I don't know how much does it affect the incorporation of the probes.

Have you ever had problem getting good incorporation because of the DNA quality?

I noticed that you use lower concentration of the dNTPs than I do, how vital do you think that might be?

Andrea,

Pretreatment of slides generally involves RNase treatment, Pepsin treatment 5-10 minutes, formaldehyde/Mgcl2 fix and then dehydration in ethanol series. Washes with 2X SSC and 1X PBS follows the treatment wherever necessary for equilibration.

For Post-hybridization washes I do 2 times 10 minutes washes in 50% formamide in 2X SSC at 37 C, and then blocking with TNT buffer, TNB buffer, 5% BSA, and detection with anti-dig and avidin antibodies with 3 cascades for each labels, then dehydration and air drying before mounting with DAPI antifade.

Dear Sirjan,

I don't know how is your porblem now. Seeing your question, the porblem might be related to your DNA isolation. In case, during isoltaion of BAC DNA you may have various E. coli contamination. In that case, although you label the same amount of DNA the proportion of your BAC might vary between the different batches leading to singal variation. Because of this problem, similar to Andrea, we  use a plasmid isolation kit (Roche in our case) that results in a relatively low yield with very high purity. We do measure the DNA and have about 2-4 ug DNA from each 10 ml grow up batch.

Thank you Szuhai,

My problem is less severe now. I have completed my experiments and the degree. Thank you for the suggestions and a lot the times that would be the case, however, contamination might not have been my issue because I tried using the kit and the use of the kit did not give me any different result. My issue was with the length of incubation time and enzyme I was using. I needed slightly longer incubation to accomplish better incorporation. Slowing down the reaction helps in some cases.

Dear Sirjan,

Sorry to answer so late but this answer could help others. Of course the quality of your extraction impact the quality of your labeling. In my experience the Life technologies kit is the best for high quantity and quality of BAC extraction. I use the maxiprep kit with small modifications (you have to increase the buffer volume of resupension, lysis and precipitation buffers twice regarding the maxiprep for plasmids and elute with the elution buffer pre-heated at 65°C). For labeling the Nick translation kit from abbott molecular, is the best (we've tried a kreatech technique that is awful! just as their probes!) and you have to perform the labeling during 16hours at 15°C. I can give the exact refernces of all products I use if someone's interested.

Good luck for your project

Best regards

Dear Elodie,

Can you send me your detailed protocols for BAC DNA preparation, nick translation probe labeling, and in situ hybridization?

Thanks!

Guangping Chen

Emory University

Elodie Laharanne

Dear Elodie, could you also send me a copy of your detailed protocols just like requested by Guangping? Many Thanks

I used the Roche nick translation mix to label my bac DNA 1ug for 90min at 15 Celcius, but did not get any DNA band. I am afraid the incubation time should be increased.