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Cloning purified PCR products eluted in Qiagen's EB


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11 replies to this topic

#1 kmkocot

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Posted 31 January 2009 - 10:26 AM

Hi all,

I gel extract and purify my PCR products using Qiagen's Qiaquick kit and elute with 30 ul of elution buffer (EB). How much can I concentrate this eluted DNA in EB using a vacufuge before the salts in the buffer will negatively affect the ligation reaction?

Thanks!
Kevin

#2 phage434

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Posted 31 January 2009 - 10:35 AM

EB is 10 mM tris-HCl. If you concentrate 10x and then use 1 ul in a 10 ul ligation, you will be adding 10 mM tris-HCl to the buffer, which probably won't make much of a difference. Usually you don't want high DNA concentrations for ligation, so I'm not sure why you would want to do this. High concentrations favor long concatamers rather than recircularization, reducing the DNA fragments that will actually transform.

#3 T C

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Posted 31 January 2009 - 08:18 PM

Hey

I use freshly autoclaved water to elute, saves me from all these problems and the DNA is also stable. Atleast for 5 years at -20 degrees.

Best
TC

#4 phage434

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Posted 31 January 2009 - 08:42 PM

Elution depends on the pH of the elution buffer, which should be slightly alkaline. Pure water has an uncontrolled pH, tending to the acidic as it absorbs CO2 from the atmosphere. You could dilute the 10 mM Tris to 1 mM, and still control the pH effectively.

#5 kmkocot

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Posted 02 February 2009 - 02:45 PM

Thank you for the replies. I have a few PCR products that produced faint bands that I gel purified. I am using Promega's pGEM-T cloning kit so I want to use as much of my product for the ligation as I can.

#6 tyrael

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Posted 12 February 2009 - 05:56 AM

i was using Vivantis GF-1 Nucleic Acid Extraction Kit to purify my DNA.....
however i was in a dilemma during the last step, i.e.: elution of DNA..

i was supposed to use the EB (elution buffer) to elute, however, the sequencing company that i would send to, request me to store my purified DNA in water.

so which one does matter ? as mentioned above, water might have uncontrolled pH, while EB has slightly alkaline pH...

any comments, pls ?
thank you.

#7 Kami23

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Posted 12 February 2009 - 06:02 AM

i was using Vivantis GF-1 Nucleic Acid Extraction Kit to purify my DNA.....
however i was in a dilemma during the last step, i.e.: elution of DNA..

i was supposed to use the EB (elution buffer) to elute, however, the sequencing company that i would send to, request me to store my purified DNA in water.

so which one does matter ? as mentioned above, water might have uncontrolled pH, while EB has slightly alkaline pH...

any comments, pls ?
thank you.



I have always used water to be on the safe side and have had some really nice results. And surely the content of CO2 would be low in autoclaved water and therefore wont stay dissolved?

Edited by Kami23, 12 February 2009 - 06:03 AM.


#8 perneseblue

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Posted 12 February 2009 - 06:13 AM

i was using Vivantis GF-1 Nucleic Acid Extraction Kit to purify my DNA.....
however i was in a dilemma during the last step, i.e.: elution of DNA..

i was supposed to use the EB (elution buffer) to elute, however, the sequencing company that i would send to, request me to store my purified DNA in water.

so which one does matter ? as mentioned above, water might have uncontrolled pH, while EB has slightly alkaline pH...

any comments, pls ?
thank you.



I have always used water to be on the safe side and have had some really nice results. And surely the content of CO2 would be low in autoclaved water and therefore wont stay dissolved?


actually due to the pressure and lack of buffering, freshly autoclave is a little acidic around pH5.
Water which has time to equilibriate with the atmosphere has a pH of around 5.6.
May your PCR products be long, your protocols short and your boss on holiday

#9 tyrael

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Posted 12 February 2009 - 07:45 AM

if that so, autoclaved distilled water is stil slightly acidic....
hmm, i better do a pH value check before i elute it with water.

DNA stored in EB wil last longer than water ? or it's the same ?

#10 perneseblue

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Posted 12 February 2009 - 09:28 AM

if that so, autoclaved distilled water is stil slightly acidic....
hmm, i better do a pH value check before i elute it with water.

DNA stored in EB wil last longer than water ? or it's the same ?


DNA in EB would probably last longer as it contains tris-HCl buffer. How much longer would the prep be useful is dependent on how much DNA was present initially and is it ssDNA or dsDNA.
May your PCR products be long, your protocols short and your boss on holiday

#11 WHR

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Posted 15 February 2009 - 09:08 PM

Thank you for the replies. I have a few PCR products that produced faint bands that I gel purified. I am using Promega's pGEM-T cloning kit so I want to use as much of my product for the ligation as I can.



hi,
TA cloning is easy. I think a visible band of DNA is sufficient for cloning. You don't need to concentrate it.
If you want to, a NaOAc/2-propanol precipitation can do the job.

#12 little mouse

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Posted 23 February 2009 - 07:12 AM

i was using Vivantis GF-1 Nucleic Acid Extraction Kit to purify my DNA.....
however i was in a dilemma during the last step, i.e.: elution of DNA..

i was supposed to use the EB (elution buffer) to elute, however, the sequencing company that i would send to, request me to store my purified DNA in water.

so which one does matter ? as mentioned above, water might have uncontrolled pH, while EB has slightly alkaline pH...

any comments, pls ?
thank you.


I elute in TE, and dilute at least 20 times in water to sequence.
you could elute in tris buffer (EDTA will inhibit the PCR, unless it is enough diluted), and prepare the dilution for sequencing in water.




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