how does dsDNA probes work in Northern ? - its a very basic qn but i am confused (Apr/18/2006 )

hello all

i know its a very basic qn ,
i have to prepare a DNA probe for Northern
my lab mate suggested ds DNA
the suggestions were
1)
get plasmid with the insert gene , digest it with Restriction enzyme to digest the gene fragment
elute the DNA from gel
check concentration etc
and go for probe making
take around 50ng of DNA
denaturation by keeping at 95-100 degree in water bath
adding the mixutre of :
specific forward primer of the gene
klenow fragment
buffer and dATP ,dGTP ,dTTPand p32dCTP

and incubating at 37 degree for aroudn 30 min

2) also PCR fragment of gene
purified and used in the above reaction instead of eluted gene fragment


how will a doulbe stranded specific gene fragment bind with the specific RNA in the membrane??

isnt that this above protocol only works for random primers in atleat 1-2Kb DNa ?

after thinking about it i think i should take more than just the gene fragment from the plasmid , or even the whole plasmid and use the specific primer in the reaction to get a good binding

will it work?

is it true that single stranded DNA probes are better than ds DNA probes ?
and why?


any thought appreciated

thank you in advance

ds DNA is denatured at boiling and cooled in ice. That'll form ss DNA if the concentration is appropriate. Well, one strand will hybridize to the RNA molecule, i.e. the antisense strand while the other won't.

choice between ss and ds DNA won't make any marked differences in getting the signal you want.

hi
rinciple of such kits if to do some nicks in dna and by elongation, replace almost all nucleotides.
CTP is incorporated and replace most of the C in your sequence. how? it's because the number of p32 CTP added in reaction is in large excess in regard of the "cold" CTP from the sequence to label.

hello

thank you for the explanation

so will it mean that i can take just my gene fragment as Dna template
or should i keep the whole plasmid?/

best is cDNA