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why i couldn't get result for my northern.help! - (Jan/12/2008 )

i'm doing my work on capsaicin synthase, gene encoded for pungency factor in pepper.
i already isolate that gene from chilli placenta and now are going to look for the gene expression...
i've done my northern twice but still couldn't get any result...
according to the previous article i supposely can get different level of expression from different fruit stages or at least get expression in placenta compared to the other organs.
i've follow the protocol exactly for pre-hyb,hyb and also prob labelling..but still couldn't get the band..my film was clear without any traces or band..
is it becoz there were no expression or the expression level for that gene was not good??
can anyone please help me... :unsure:

-belle-

well at this point it's quite hard to give definintive answer, but...
did you check the specificity of your probe in terms of sequence, and also can you obtain a probe with higher tm ?
Maybe you may consider the fact to evaluate random labelling and T4PNK labelling
did you check temperatures and stringency ? i got better eresutls using church buffer rather than denhardt's buffer (also it was the contrary for other gene...)
you can make a small 15% TBE-PAGE gel and load 0?5┬Ál of your probe to see activity of it.
do you have a possibility of a positive control ?

you can expose your film with reducing washing times and wah at room temperature.
If signal is too loud, you can was more.

-fred_33-

A good strategy in cases like this is to work backwards. Make sure you can detect the probe with the film. Do serial dilutions of the probe and determine how much probe is necessary to detect. If necessary, optimize your detection.

Move on to hybridizing. Start with a positive sample. You already have DNA, so do serial dilutions of the DNA, spot on a membrane, and hybridize with your probe. Determine how much DNA is necessary.

Move on to RNA. Isolate total RNA and spot serial dilutions. Detect with your probe, and determine the minimal amount of RNA. Optimize the hybridization conditions here to maximize sensitivity (you may want a negative RNA control, such as RNA from a species not expressing the gene).

Finally move on to Northerns, which should, at this point, "just work." I find it useful to continue spotting probe and positive RNA control dilutions onto membranes even after things are working.

-phage434-

i've already try to detect my probe with the film and it was ok..i can see the band from 100ng of my DNA.
So it means that, my probe was ok..so i will try to do serial dilution using my total RNA and also for negative control to determine how much RNA i need for detection..

and i just want to know, if i use the probe from the same plant (chilli) but test on RNA from different varieties, is it effect my result??

QUOTE (phage434 @ Jan 13 2008, 11:31 AM)
A good strategy in cases like this is to work backwards. Make sure you can detect the probe with the film. Do serial dilutions of the probe and determine how much probe is necessary to detect. If necessary, optimize your detection.

Move on to hybridizing. Start with a positive sample. You already have DNA, so do serial dilutions of the DNA, spot on a membrane, and hybridize with your probe. Determine how much DNA is necessary.

Move on to RNA. Isolate total RNA and spot serial dilutions. Detect with your probe, and determine the minimal amount of RNA. Optimize the hybridization conditions here to maximize sensitivity (you may want a negative RNA control, such as RNA from a species not expressing the gene).

Finally move on to Northerns, which should, at this point, "just work." I find it useful to continue spotting probe and positive RNA control dilutions onto membranes even after things are working.

-belle-