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PCR smearing and...problems!


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#1 jamestoon1

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Posted 22 March 2012 - 12:55 AM

My PCR used to be like this:
http://desmond.image....jpg&res=medium

But now it has become like this:
http://desmond.image....jpg&res=medium

I did not change the PCR condition (temperature, duration etc) or volume (concentration) of reagents/template DNA used.
Can anyone help to figure out what is happening?
p/s: all the six lanes are from six different samples

#2 Astilius

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Posted 22 March 2012 - 01:33 AM

Has anything changed? Even just lot numbers of reagents, new tubes of same lot number reagents?
Or have things not changed? Have you been using the same tube of dNTPs or primers for a long time?
To the last, I grapple with thee; from Hell's heart, I stab at thee; for hate's sake, I spit my last breath at thee.

#3 jamestoon1

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Posted 22 March 2012 - 07:50 AM

Nothing has been changed.
I am using Phusion PCR Master Mix.
I obtain results similar to the first gel picture for the first 3 or 4 times I use the reagent. And then after that, all my results were similar to the second gel picture. I tried to use other tubes of master mix of the same lot number, and I still get results similar to the 2nd gel shown above.

#4 hobglobin

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Posted 22 March 2012 - 10:28 AM

perhaps DNA quality changed (e.g. slowly degradation)? if you have earlier templates (frozen) that worked well you should try out them...
check also the thermal cycler, sometimes the program was changed by somebody...

Edited by hobglobin, 22 March 2012 - 10:38 AM.

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#5 Astilius

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Posted 23 March 2012 - 12:50 AM

Also, what about water? Get a new bottle of nulease free water, your current one might be contaminated.
To the last, I grapple with thee; from Hell's heart, I stab at thee; for hate's sake, I spit my last breath at thee.

#6 jamestoon1

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Posted 23 March 2012 - 05:19 AM

perhaps DNA quality changed (e.g. slowly degradation)? if you have earlier templates (frozen) that worked well you should try out them...
check also the thermal cycler, sometimes the program was changed by somebody...

i have checked the thermal cycler program, it is fine. it is possible that dna quality has changed, because the experiment i am doing is actually a continuation of previous study, so i am using dna which has been stored for some time.
the dna is very precious (limited), we no longer have dna from the same study subject, so my question now is, with degraded dna, what can i do to obtain a discrete band of expected sizE?

#7 jamestoon1

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Posted 23 March 2012 - 05:20 AM

Also, what about water? Get a new bottle of nulease free water, your current one might be contaminated.

not the water. because the same tube of water can produce good result with other samples. :)

#8 Astilius

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Posted 23 March 2012 - 05:39 AM


perhaps DNA quality changed (e.g. slowly degradation)? if you have earlier templates (frozen) that worked well you should try out them...
check also the thermal cycler, sometimes the program was changed by somebody...

i have checked the thermal cycler program, it is fine. it is possible that dna quality has changed, because the experiment i am doing is actually a continuation of previous study, so i am using dna which has been stored for some time.
the dna is very precious (limited), we no longer have dna from the same study subject, so my question now is, with degraded dna, what can i do to obtain a discrete band of expected sizE?


Ah, the DNA it is then. There's nothing you can do with this sample of DNA.
For future reference you should look at better ways of handling sample DNA. If you're multiply freeze-thawing DNA then don't, split samples into multiple small aliquots and freeze them all so that each sample goes through as few such cycles as possible. Freeze as cold as you can achieve and keep samples stable. Store in an appropriate buffer.
But for this sample there's not much you can do if the DNA is, effectively, destroyed. There's no rewind on entropy.
To the last, I grapple with thee; from Hell's heart, I stab at thee; for hate's sake, I spit my last breath at thee.




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