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No DNA after PCR product purification

PCR product purification

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9 replies to this topic

#1 tamarine

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Posted 26 May 2013 - 11:54 PM

Hi expertises..,


I wonder if somebody here could help me with the mess..

Currently, i am working with the identification of specific bacteria, employing the specific primer.
subsequently, i have to make sure the product generated with the sequencing platform. surely, i got the target band in a quite intensity.

Here the problem begins.,
Once i purify the PCR product using PCR product purification kit (Geneaid), no product detected. I completely was followed the protocol of the kit. previously, i purified the 16S band with the same kit, and the result was just fine.

any idea what the matter with my experiment..?


Thanks in advance

#2 Curtis

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Posted 27 May 2013 - 12:08 AM

it happens. Some times we get 10 ng of purified product. we can't say it doesn't exist. purify again, use fresh binding buffer. when you add water or TE buffer in the end make sure you keep for at least a minute to dissolve DNA before spin .

#3 tamarine

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Posted 27 May 2013 - 08:01 AM

Hi Curtis,

Thank you for the suggestion

i got the hint. seems i should use the newest buffer binding. since, actually the newest one is available. this will be my first remedy for tomorrow work.

i was already let the elution (water / TE) for 5 mins before spinning. Or perhaps, i made a mistake by adding abundant elution into it..? i added 20 microliters. what if i lessen it to just 10 micro ?

#4 tamarine

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Posted 27 May 2013 - 08:04 AM

umh, almost forgotten..

would be ok, if i load the so-called-unexist band into sequencing..? let it say, sort of trial..
What do you think..?

#5 phage434

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Posted 27 May 2013 - 12:27 PM

So, if I understand you correctly, you run a gel with a pcr product (before purification) and see a band. Then, you purify it with a column cleanup, and run the result on a gel, and the band is gone. Is that right? What kind of kit is Geneaid, I'm not familiar with it. Did you make sure to add ethanol to the wash buffer?

#6 tamarine

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Posted 27 May 2013 - 08:43 PM

HI Phage,..

Yes, you got it right. Geneaid is basically similar with other column cleanup kit. it has cheaper price, though.. (as you know, experiment cost should be maintained..Posted Image ) here is the link http://www.geneaid.c...cr-cleanup-kit.
previously, i used Qiagen kit. things worked well out. this kit is fine too beforehand. but, i didn't know what was going wrong. when i used it in my specific primer. it messed up.

today, i use the newest kit. but the result was just the same. I also try to purify the gel. it still doesn't exist.
the insane trial i have conducted too. i load the "invisible band" to sequencing work.
guess what?!..the result was awful. lol.

hope you will understand my english. it quite bad, Posted Image

need you suggestion pretty bad.

#7 tamarine

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Posted 27 May 2013 - 08:45 PM

ummh, yes i did. i washed it with ethanol. since, it's all in a package..

#8 phage434

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Posted 28 May 2013 - 05:41 AM

Most column based purification kits omit the ethanol in the wash buffer for shipment. The ethanol needs to be added before the wash buffer is used. Make absolutely certain your wash buffer has ethanol, or your DNA will be washed away.

You can trace where your DNA is going. You know there is a band of input DNA. Sample all of the flow through, wash, and elutions and run them on a gel. Somewhere, your DNA is hiding. You might want to try this with a DNA sample which is easier to produce -- the problem is likely not specific to your DNA sample.

#9 tamarine

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Posted 28 May 2013 - 10:54 PM

Most column based purification kits omit the ethanol in the wash buffer for shipment. The ethanol needs to be added before the wash buffer is used. Make absolutely certain your wash buffer has ethanol, or your DNA will be washed away.

You can trace where your DNA is going. You know there is a band of input DNA. Sample all of the flow through, wash, and elutions and run them on a gel. Somewhere, your DNA is hiding. You might want to try this with a DNA sample which is easier to produce -- the problem is likely not specific to your DNA sample.


ethanol has already added into wash buffer (my colleague did it). sorry for earlier misunderstanding.

How should i trace the hidden DNA? i don't understand clearly, should i load the binding solution and wash buffer trash onto the gel?

the generated band is not in a bright intensity. then i thought to dilute it in a less TE/water/elution volume. but, both PCR and gel clean up were still the same. not any progress revealed.
meanwhile, when i worked with 16S primer, things gone just right. i had no problems in a pcr purification stage.

#10 notjustalabtech

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Posted 09 June 2013 - 10:36 AM

Saving the "trash" or supernatants after each step and loading onto a gel would allow you to determine at which step the purification isn't working. I assume you ran your pcr on a gel prior to purification just to see if the amplification worked? Otherwise, then your problem is the pcr, not the purification. You kept mentioning that your 16s pcr purification worked, so I assume this is a different pcr protocol for a different gene?

So make sure you save all supernatants and separate them by each step in the purification. Run them on a gel (including the last step where your final product is supposed to be) and you can compare each step and see where your problem lies. If it's not in any of the steps, then your DNA is probably still in the column. Something that has always worked for me is increasing the incubation time of the wash buffer to 10 min and elution buffer on the column to 5-10min (even though the qiagen kit says 1) and I'm able to triple my yield.

Edited by notjustalabtech, 09 June 2013 - 10:36 AM.






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