Technique / Molecular Biology / DNA analysis techniques / DNA southern blot


Gene copy number by slot-blot hybridization



Gene copy number by slot-blot hybridization

I'm planning to use slot blot hybridization to calculate the gene
copy number in the genome. However, my slot blot give a very weak signal
even 50ug of genomic DNA was applied. Is there any possible casue for
this..?? I am using DIG labelling and chemiluminecence detection. The
system works fine for southern blot (using the same probe and genomic
DNA), which can detect a single copy gene using 5 - 10ug of genomic DNA.
So, I don't know what's wrong with the slot blot. Can anyone give any
suggestion??


Regards,
Lum
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Are you denaturing your genomic DNA before you apply it to the filter?
Colin Rasmussen
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Yes, by adding NaOH to final conc. of 0.4N and boil for 16 minutes.
However, after the slot blot process, I notice something stick on the
membrane. This is only observed in slot with high amount of DNA (e.g. 35ug) Is
that the DNA..?? And how should I fix the DNA on the membrane??


Regards,
Lum
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Hi,


How do you fix the DNA on the membrane? Sometimes, just drying the membrane
would be enough; but if you want a better fixation you may try to use UV
irradiation or heat fixation (placing the membrane in an incubator at 80ºC for
1.5-2 hours would do both a partial denaturation of the DNA and fixation to the
membrane).
regards, Llu�-s
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I've try both UV-fixation and baking at 120C for 40 mins. Both of them have
similar result.


Regards,
Lum
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just try placing the nylong membrane after transfer in a microwave oven at
full setting (atleast 800W) for 2.45 min. This came in a NAR paper around
1991. I don't remember the exact reference, but it gives excellent fixing
and much better signals than UV crosslinking or hot oven fixing at 80C,
try this out.
bye
cheers
jakku
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Why would you add that amount of NaOH AND boil. Sounds pretty harsh to me. I'd
simply boil it for 10 minutes, cool in ice, and then apply to the blotter.
Colin
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> I'm planning to use slot blot hybridization to calculate the gene
> copy number in the genome. However, my slot blot give a very weak signal
> even 50ug of genomic DNA was applied. Is there any possible casue for
> this..?? I am using DIG labelling and chemiluminecence detection. The
> system works fine for southern blot (using the same probe and genomic
> DNA), which can detect a single copy gene using 5 - 10ug of genomic DNA.
> So, I don't know what's wrong with the slot blot. Can anyone give any
> suggestion??


I battled with the same problem trying to determine copy number in
transgenic animals.


Let me guess are you looking at mammalian genome? If so I suspect
that this approach will not work :-=(. There is too much negative DNA
in the way with large genomes.; the probe simply cannot find the
target. I eventually concluded that Southerns were the only way until
I could persuade someone to buy me a Quantitative PCR machine.


If you do find a solution please post it, it would be very useful
David
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Not true. I've done it using tissue culture cells transformed with a
plasmid, and I wanted to determine the copy number of the plasmid. One thing
that might also help is to RE digest the genomic DNA prior to denaturation
and blotting.
Colin
Newsgroup archive


Last update 17-Jan-2002, Rating of 4 votes.

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By Ranadheer on 10-Apr-2008
Please suggest me a protocol for the determination of gene copy number by Realtime PCR
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By archana on 16-Aug-2007
i want to know the copy no of a specific gene in plant genome.what appoarch should i follow?
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By senthil on 15-Feb-2005
my question is that how to crosslink a plasmid to a protein? iwant a protocol of uv crosslinking using a ordinary uv light
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By vivek on 12-Jan-2005
hai
discussion is nice,
i am doing my masters project in molecular biology,
can any one give the detail description how the prehybridisation and hybridisation of DNA with the labeeled probe,

hope i will get it,
'
bye
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By Shalini Sharma on 28-Apr-2004
i am looking for a protocol for determination of gene copy number in a transfected mammalian cell line,where amplification was done using MTX.I know that southern blot analysis can be used but can not figure out how copy number is to be calculated .If anyone can help,please.
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By tom on 16-Feb-2004
I have some problems with determine the insert copy number using the southern blot method. I don't get any signal with 4 different probes and I use 10ug of DNA. However, I have a very strong signal in the controls, plasmid DNA. So the protocol works. Could someone please help me

tom
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By krismenon on 01-Apr-2003
I am looking for a protocol to determine the gene copy number in a transfectant cell line which has been subjected to gene amplification on MTX(Dhfr mediated).



Could anyone give me feedbacks on this please.
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By Tracy on 17-Jan-2002
Hi,

I was wondering if you'd be able to give some advise. I'm an inexperienced molecular biology student..I'm trying to do a Southern to determine copy number. I will be using a DIG labelling kit to make my probe and chemiluminescence for detection. But before I start, how do I optimize the kit ie: determine how much genomic DNA I need to load to produce a readable signal?


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