6 replies to this topic

[MPK]

Hi everyone,

I posted the following question elswhere but i hadn't no response yet ! so, i post it here hoping obtain your opinions !!

I'd like to know if Salmon Sperm DNA is really necessary in the DNA/RNA prehybridization procedures? what happens if i don't use it? and when it's used, does it affect the hybridization efficiency while excercing a competitive role with the specific sequence and diminish the good match probe/target? Does it come down to cover the DNA and make it hardly accessible to be hybridized with it's complementary strand?

ThankU

[DevGrp]

I must admit when doing southerns I've frequently left it out without any major background problems. It's usually added to fill up the membrane to whcih DNA hasn't already bound, so that your probe doesn't stick to the membrane in a non-specific way (dired milk / BSA / gelatin is used for a similar reason in westerns)
I have found using Denharts or Church  pre-hybridisation buffer without salmon (or herring) sperm DNA works just as well.
Ian

[littlecell]

when do northern blot i used the perfectHyb plus hybridization buffer (Sigma, H7033), which worked very well and can be used as prehybridization and hybridization buffer as well.

[MPK]

Thank you my friends DevGrp and littlecell  !!

[seahow]

i think the that salmon sperm DNA with proper length  is required in your northern blot; but need not in sourthern blot.

beacause in northern blot what you will detect is mRNA which has 18 s 28s rRNA . the rRNA can hybride with any probe for its homolous.



seahow

[seenew]

I use a kit from Amersham, in the protocol it said that  salmon sperm DNA is not necessary. if you add, it will decrease background, but it will also decrease hybridization effiency.

[luminb]

I would say that prehyb with ssDNA is necessary to make your film cleaner and prettier. As if you don't use it, it will still give bands but the whole blot is darker. I never found any problems that prehyb will decrease the binding efficiency of your probe. I use quickhyb from stratagen and usually it takes only half hour for prehyb and 1-2 hours for hyb.