Sothern hybridization with digoxin-labeled probes - (Jul/22/2008 )
I am wringting to consult from those who are experienced in Sothern hybridization with digoxin-labeled probes . The purpose is to detct the copy number of transgenes in Arabisopsis. How many genomic DNA should be used and how many the probe? I plan to use 35S or NPTII sequence as the probe. I have experiences in Sothern hybridization using P-32 labled probes, but what should be noticed when use digoxin-labeled probes ?
Thank you very much!
DIG southerns are very similar to radio-labeled. The amount of DNA you need will be determined by the abundance of the target sequence, for example, if your target has only one copy per genome then you will need more DNA than if your target has 50 copies per genome. Having said that, as you are looking to see how many copies per genome you have you will need a reasonable amount of DNA. I would start with 1 microgram and test from there in a dilution series of 1, 2, 5, 10, 15, 20 micrograms. I have typically found that 5 ug is enough for single copy detection in human DNA using DIG labeled probes
I can recommend PCR labeling as the signal is much stronger than end-labeling, because you have typically several DIG labeled UTPs per probe rather than just one. DIG probes are heavier when run on a gel due to the size of the DIG. The DIG manual from Roche is really good and has trouble shooting for almost every step.
it is really nice of you. thanks a lot!