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Problem with DNA extraction from 1% agarose Gel


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#1 dgclone

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Posted 02 June 2009 - 09:47 AM

I'm a little lost here and hoping that someone may be able to shed some light on a problem I'm having.

I'm trying to purify and isolate a PCR fragment (~1.8kb) by running about 10 - 20ul of my PCR product on a 1% agarose gel 110V ~15mins. So far I've tried three methods, Qiaex 2 gel extraction kit, Qiagen Qiaquick Gel Extraction kit, and using DAE81 filter paper to purify my DNA. In both Qiagen kits I get no yield after following the protocol exactly. The only variables are the length of time I dry the pellet in the Qiaex 2 kit and the elution buffer ie ddH20 pH 8 or Tris-HCL pH 8.

I've had previous issues with the elution buffer and have checked that twice now. I have a very strong PCR product band in the ug yield and even have tried diluting the PCR product in case the [DNA] was too high and somehow caused problems with binding. Washed and cleaned and rechecked my TAE buffer, gel, machine and reagents.

The DAE filter paper method gives me aprox 30% yield but as I'm attempting to do cloning after restriction digest and purification again my [DNA] is too low for ligation.

I'm attempting to use a new kit from another lab at the moment but if anyone else has had similar problems or any insight I would very much appreciate the help!! Thanks,

#2 xyz

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Posted 02 June 2009 - 10:00 AM

Hi,
I have tried with qiagen Qiaquick kit and it didn,t give me good yield (how dou check the yield?) So I was being suggested to use Qiaes2 kit. So i will do with that. so 1 one question I have is that in both of kits elution buffer are same or different? according to me its same 10 mMTris Cl PH 8.0, but as u said that both use different buffer?

I'm a little lost here and hoping that someone may be able to shed some light on a problem I'm having.

I'm trying to purify and isolate a PCR fragment (~1.8kb) by running about 10 - 20ul of my PCR product on a 1% agarose gel 110V ~15mins. So far I've tried three methods, Qiaex 2 gel extraction kit, Qiagen Qiaquick Gel Extraction kit, and using DAE81 filter paper to purify my DNA. In both Qiagen kits I get no yield after following the protocol exactly. The only variables are the length of time I dry the pellet in the Qiaex 2 kit and the elution buffer ie ddH20 pH 8 or Tris-HCL pH 8.

I've had previous issues with the elution buffer and have checked that twice now. I have a very strong PCR product band in the ug yield and even have tried diluting the PCR product in case the [DNA] was too high and somehow caused problems with binding. Washed and cleaned and rechecked my TAE buffer, gel, machine and reagents.

The DAE filter paper method gives me aprox 30% yield but as I'm attempting to do cloning after restriction digest and purification again my [DNA] is too low for ligation.

I'm attempting to use a new kit from another lab at the moment but if anyone else has had similar problems or any insight I would very much appreciate the help!! Thanks,



#3 microgirl

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Posted 02 June 2009 - 10:31 AM

I get horrible yields with the Qiagen kits. However. . . what are you trying to do with your DNA afterwards? I have had no problems taking the negative yields of DNA gotten from the Qiagen kits and ligating it into vectors and transforming with it. Plenty of colonies.

#4 dgclone

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Posted 02 June 2009 - 10:33 AM

Hi there,
I checked the yield by comparison to my ladder with a known concentration. Further I ran a gel of the purified product vs. the PCR product and IF I see a band it's at least 100 - 1000X more dilute than my PCR product ie to low to use for RE digest and then purify again for transformation.

As per the elution buffers they are the same for both kits but the it mentions using ddH20 pH 7 - 8.5 or the EB provided or your own Tris-HCl buffer pH 8. I've tried all of them with no luck with both kits. I'm beginning to suspect contamination with my QX1 buffers etc as even with nuclease/contamination present I would expect to get at least some DNA or streaks even with my purified sample.

Hi,
I have tried with qiagen Qiaquick kit and it didn,t give me good yield (how dou check the yield?) So I was being suggested to use Qiaes2 kit. So i will do with that. so 1 one question I have is that in both of kits elution buffer are same or different? according to me its same 10 mMTris Cl PH 8.0, but as u said that both use different buffer?

I'm a little lost here and hoping that someone may be able to shed some light on a problem I'm having.

I'm trying to purify and isolate a PCR fragment (~1.8kb) by running about 10 - 20ul of my PCR product on a 1% agarose gel 110V ~15mins. So far I've tried three methods, Qiaex 2 gel extraction kit, Qiagen Qiaquick Gel Extraction kit, and using DAE81 filter paper to purify my DNA. In both Qiagen kits I get no yield after following the protocol exactly. The only variables are the length of time I dry the pellet in the Qiaex 2 kit and the elution buffer ie ddH20 pH 8 or Tris-HCL pH 8.

I've had previous issues with the elution buffer and have checked that twice now. I have a very strong PCR product band in the ug yield and even have tried diluting the PCR product in case the [DNA] was too high and somehow caused problems with binding. Washed and cleaned and rechecked my TAE buffer, gel, machine and reagents.

The DAE filter paper method gives me aprox 30% yield but as I'm attempting to do cloning after restriction digest and purification again my [DNA] is too low for ligation.

I'm attempting to use a new kit from another lab at the moment but if anyone else has had similar problems or any insight I would very much appreciate the help!! Thanks,



#5 dgclone

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Posted 02 June 2009 - 12:18 PM

Hey thanks for the reply,

I'm trying to clone but it's not really working out for me as of yet.

To reiterate my PCR isn't perfectly clear, little bit of streaking +primer dimers --> hoping to purify for a RE digest by agarose gel extraction --> ligation/transformation...

I've tried taking the raw PCR product --> ethanol precipitation and then a RE digest to avoid as much loss as possible. At that point I've always done a gel to purify my cut product/check to make sure it is actually cut before ligation. I'm thinking about not even doing that now to see if I can get any positive colonies! It's not even that the I'm upset at a low yield it's just that it's not there and I haven't gotten any positive colonies trying a 1:1, 1:2, 1:3, 1:4 1:5 vector to insert ratio.


I get horrible yields with the Qiagen kits. However. . . what are you trying to do with your DNA afterwards? I have had no problems taking the negative yields of DNA gotten from the Qiagen kits and ligating it into vectors and transforming with it. Plenty of colonies.



#6 AquaPlasmid

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Posted 03 June 2009 - 07:28 AM

No pun inteneded, but I am amazed at so many of you wasting money on gel purification kits (I think I have posted on this topic many times). For daily cloning, you could just run a low-melting gel in TAE, after cutting out the band (don't expose it to UV light by using side-by-side wells, one lane for UV, the other for recovery), put it in a spin filter, melt the gel at 70*C, freeze it at -20 or 70*C, then spin at room temp, and you've got the DNA fragment for ligation. For library cloning, you add b-agarase to the melted gel equilibrated at 45*C and incubate for a hour to digest the agarose gel slice.

Aqua
www.aquaplasmid.com


#7 xyz

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Posted 03 June 2009 - 07:39 AM

There might be some contamination in buffers!!!

I get horrible yields with the Qiagen kits. However. . . what are you trying to do with your DNA afterwards? I have had no problems taking the negative yields of DNA gotten from the Qiagen kits and ligating it into vectors and transforming with it. Plenty of colonies.



#8 microgirl

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Posted 03 June 2009 - 09:40 AM

Why don't you just try running it through a PCR clean-up kit rather than gel extracting. This will remove primers, primer dimers, enzymes, salts, etc. The restriction digest/ligation should (rather than TA cloning) should also help you to get the right piece into your vector.

Hey thanks for the reply,

I'm trying to clone but it's not really working out for me as of yet.

To reiterate my PCR isn't perfectly clear, little bit of streaking +primer dimers --> hoping to purify for a RE digest by agarose gel extraction --> ligation/transformation...

I've tried taking the raw PCR product --> ethanol precipitation and then a RE digest to avoid as much loss as possible. At that point I've always done a gel to purify my cut product/check to make sure it is actually cut before ligation. I'm thinking about not even doing that now to see if I can get any positive colonies! It's not even that the I'm upset at a low yield it's just that it's not there and I haven't gotten any positive colonies trying a 1:1, 1:2, 1:3, 1:4 1:5 vector to insert ratio.


I get horrible yields with the Qiagen kits. However. . . what are you trying to do with your DNA afterwards? I have had no problems taking the negative yields of DNA gotten from the Qiagen kits and ligating it into vectors and transforming with it. Plenty of colonies.



#9 georgiadave

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Posted 05 June 2009 - 10:02 PM

Reduce the amount of original buffer volume that you use. If I remember correctly they say to use 5 volumes?...use as few as possible.

Also, only do post staining so the DNA doesn't have EtBr since this is an obvious problem for cloning.

Check the lot number on the columns...they only last UP TO one year...if they don't say manufacture date call tech support.

If your columns turn out to be 'old' then there is a way to 'recharge' them. Pipet 0.1ml 4M NaOH onto the membrane and spin for 30 sec at max speed. This will not damage your DNA. After emptying the collection tube resume your extraction...there is no need to clean the membrane.

Preheat elution buffer @ 60deg C

#10 jiajia1987

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Posted 07 June 2009 - 05:01 PM

I've never had any problems performing DNA extraction from 1% agarose gel with QIAGEN kits. The only time when I had a problem was when I used a PE buffer without any ethanol, and that lead to elution of all the DNA during the washing steps, leaving me with no DNA at the end of the procedure. I always use water to elute my DNA. If there are no problems with the composition of your buffers, then contamination could be a problem. My friends do not have a problem with the kits too.




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