Hello,
If I have ~40kb sheared metagenomic DNA sample, melt it at 95 C, and then bring it back to room temperature, would you expect it to anneal back together and thus become visible with a dsDNA dye? If no, would you expect this to work for a 40kb sample of identical DNA?
I'm asking because I'm wondering why a template band isn't visible after doing a PCR reaction, when such a band would be visible had the sample not gone into the thermocycler.
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#2
Ameya P
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Posted 20 October 2010 - 08:59 PM
JP_, on 20 October 2010 - 01:03 PM, said:
Hello,
If I have ~40kb sheared metagenomic DNA sample, melt it at 95 C, and then bring it back to room temperature, would you expect it to anneal back together and thus become visible with a dsDNA dye? If no, would you expect this to work for a 40kb sample of identical DNA?
I'm asking because I'm wondering why a template band isn't visible after doing a PCR reaction, when such a band would be visible had the sample not gone into the thermocycler.
If I have ~40kb sheared metagenomic DNA sample, melt it at 95 C, and then bring it back to room temperature, would you expect it to anneal back together and thus become visible with a dsDNA dye? If no, would you expect this to work for a 40kb sample of identical DNA?
I'm asking because I'm wondering why a template band isn't visible after doing a PCR reaction, when such a band would be visible had the sample not gone into the thermocycler.
I think the template is not seen on the gel, once you put it into a reaction tube, is cause its getting diluted (say 20 times).
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JP_
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Posted 21 October 2010 - 10:26 AM
gt_ameya, on 20 October 2010 - 08:59 PM, said:
I think the template is not seen on the gel, once you put it into a reaction tube, is cause its getting diluted (say 20 times).
Yeah I am taking that into account - in spite of the dilution, I'm putting an equal amount of template DNA into both wells - only difference is one template DNA went through PCR (with mastermix, primers, etc), and the other did not (I just diluted it in some water to load the gel). So wondering if this is normal (the 40kb templates won't be expected to anneal), or indicates a problem like template DNA being degraded in PCR.
This PCR reaction was not successful btw - currently troubleshooting it, and looking at the lack of the template band as a potential clue.
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chromatin
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Posted 21 October 2010 - 07:15 PM
It may not anneal perfectly, and not go into the gel. Additionally, it may get sheared (become shorter fragments) due to hydrodynamic forces. Same reason DNA breaks during vortexing.
I hope my suggestions make sense.
http://www.bioprotocols.info/
I hope my suggestions make sense.
http://www.bioprotocols.info/
#5
leelee
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Posted 21 October 2010 - 08:38 PM
You are saying that the amount of template you add to your PCR reaction could be visualised on a gel right?
I would say that is way too much template and may be why your PCR is failing. First thing I would do to trouble shoot your PCR would be to do a dilution series of your template and PCR on those- and see what happens. I'd start with 1/10, 1/100, 1/1000 and 1/5000
I would say that is way too much template and may be why your PCR is failing. First thing I would do to trouble shoot your PCR would be to do a dilution series of your template and PCR on those- and see what happens. I'd start with 1/10, 1/100, 1/1000 and 1/5000
