ghost band in PCR? - (Oct/13/2008 )
Hi ,everyone!
I got a promble in recent trials.We were making use of RFLP to differentiate the SNP of a extron.When we did the PCR ,we got the expected band ,about 500 bp,while a slightly samller band ahead of it in gel,seeming 30-40 bp smaller.But the marker is ok. 
Further more, when we applied restriction enzyme to the product, the ghost disappeared! We got excatly the result expacted. So what's it?Appreciation to any suggestion!
-shoes-
QUOTE (shoes @ Oct 13 2008, 05:32 AM)
Hi ,everyone!
I got a promble in recent trials.We were making use of RFLP to differentiate the SNP of a extron.When we did the PCR ,we got the expected band ,about 500 bp,while a slightly samller band ahead of it in gel,seeming 30-40 bp smaller.But the marker is ok.
Further more, when we applied restriction enzyme to the product, the ghost disappeared! We got excatly the result expacted. So what's it?Appreciation to any suggestion!
I got a promble in recent trials.We were making use of RFLP to differentiate the SNP of a extron.When we did the PCR ,we got the expected band ,about 500 bp,while a slightly samller band ahead of it in gel,seeming 30-40 bp smaller.But the marker is ok.
Further more, when we applied restriction enzyme to the product, the ghost disappeared!
We got excatly the result expacted. So what's it?Appreciation to any suggestion!Is the ghost band faint or quite pronounce?
got gel picture?
maybe the digested ghost band is still there as well but just harder to see because you reduce the amount of DNA used for restriction digest?
This is an odd one nevertheless.
-Hanming86-
QUOTE (Hanming86 @ Oct 14 2008, 01:53 AM)
Is the ghost band faint or quite pronounce?
got gel picture?
maybe the digested ghost band is still there as well but just harder to see because you reduce the amount of DNA used for restriction digest?
This is an odd one nevertheless.
got gel picture?
maybe the digested ghost band is still there as well but just harder to see because you reduce the amount of DNA used for restriction digest?
This is an odd one nevertheless.
the ghost was quite clear, just as the expected one!The expected digested band was clear too,cause we didn't reduce the dose.So...
Someone suggested that it might be the different helix status.A proportion of DNA might turn into supercoil while the rest remain linear.That guy met the same phenomenon when dealing with plasmid DNA, I was not.What's more, the digested product was just ok,why?
-shoes-
QUOTE (shoes @ Oct 14 2008, 07:05 AM)
QUOTE (Hanming86 @ Oct 14 2008, 01:53 AM)
Is the ghost band faint or quite pronounce?
got gel picture?
maybe the digested ghost band is still there as well but just harder to see because you reduce the amount of DNA used for restriction digest?
This is an odd one nevertheless.
got gel picture?
maybe the digested ghost band is still there as well but just harder to see because you reduce the amount of DNA used for restriction digest?
This is an odd one nevertheless.
the ghost was quite clear, just as the expected one!The expected digested band was clear too,cause we didn't reduce the dose.So...
Someone suggested that it might be the different helix status.A proportion of DNA might turn into supercoil while the rest remain linear.That guy met the same phenomenon when dealing with plasmid DNA, I was not.What's more, the digested product was just ok,why?
Can you explain what you digested with and what you mean by it was ok when it was digested?
Because the only way the ghost band dissappears is because it was degraded into small fragments so in the gel the small bands run away? Because there is no other way it can dissappear.
Did you run the gel more than once?
-veejaya-
PLasmid and PCR product is not the same to begin with
Plasmid can exist in many forms
but PCR product won't give you no supercoiled because the polymerase is not capable of such activity . Topoisomerase is the one causing supercoiling.
Perhaps it's secondary structure generated from the PCR product ( not very sure about this) but i really doubt it's supercoiling case!
-Hanming86-
QUOTE (veejaya @ Oct 15 2008, 05:55 AM)
QUOTE (shoes @ Oct 14 2008, 07:05 AM)
QUOTE (Hanming86 @ Oct 14 2008, 01:53 AM)
Is the ghost band faint or quite pronounce?
got gel picture?
maybe the digested ghost band is still there as well but just harder to see because you reduce the amount of DNA used for restriction digest?
This is an odd one nevertheless.
got gel picture?
maybe the digested ghost band is still there as well but just harder to see because you reduce the amount of DNA used for restriction digest?
This is an odd one nevertheless.
the ghost was quite clear, just as the expected one!The expected digested band was clear too,cause we didn't reduce the dose.So...
Someone suggested that it might be the different helix status.A proportion of DNA might turn into supercoil while the rest remain linear.That guy met the same phenomenon when dealing with plasmid DNA, I was not.What's more, the digested product was just ok,why?
Can you explain what you digested with and what you mean by it was ok when it was digested?
Because the only way the ghost band dissappears is because it was degraded into small fragments so in the gel the small bands run away? Because there is no other way it can dissappear.
Did you run the gel more than once?
We digested the PCR product, some of which run the electrophoresis, and the rest got digested first(or maybe I have misunderstood the meaning of "digested"?We used restriction enzyme to deal with product,what's what I what to say.)It's repeated result.
"OK" means there were no ghost bands after digested.I am not sure whether the ghost is the same sequence, with different helix status,or just different sequence.But I don't think running away fragments is the only explanation.The ghost is ran ahead of the expected product,so it should either smaller or with equivalent length.
-shoes-
has anyone made the same PCR before ? Is it possible that multiple bands are showing up because you have redundant DNA in the genome you look at ?
-ph3no-
QUOTE (ph3no @ Oct 15 2008, 10:37 AM)
has anyone made the same PCR before ? Is it possible that multiple bands are showing up because you have redundant DNA in the genome you look at ?
but if it's truly redunddant DNA, we would expect some difference in the digestion pattern. since the PCR bands differ in a 30-40 bp. my suspect is it after digestion the difference become so minute that you coundl't see it on the gel. 30-40bp becoming 15-20bp difference ( just a rough ,, very ROUGh estimation of how teh bp will distribute in the digested product.
-Hanming86-
QUOTE (ph3no @ Oct 16 2008, 02:37 AM)
has anyone made the same PCR before ? Is it possible that multiple bands are showing up because you have redundant DNA in the genome you look at ?
Someone said that to me previouly, also with the promble--where is the digested band of it?
And I was told yesterday that it might be a product-dimer(not primer-dimer ).I've never heard of that before, and have no idea how it forms.Is it possible?
-shoes-
shall I detect the sequence?or any other suggestion?
-shoes-