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Gel purification of vector DNA - low yield


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26 replies to this topic

#16 planktonica

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Posted 06 March 2009 - 03:53 PM

The Na acetate is added with the sample-GQ buffer mix. I don't add Isopropanol but the PE buffer contains Ethanol. I purified both, PCR product again and vector with the kit and got a lot of PCR and low concentration of vector again. However, it seems like 30 ng/uL is not that bad for transformation purposes.

And Swanny, I don't find your question dumb at all!

#17 ruiN

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Posted 07 March 2009 - 08:52 AM

I always warm up the elution buffer (EB, water, etc) to 60C before elution and it helps me a great deal with the concentration.

#18 jiajia1987

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Posted 10 March 2009 - 05:10 PM

I always warm up the elution buffer (EB, water, etc) to 60C before elution and it helps me a great deal with the concentration.



For me, I warm it up to 50deg before elution. The heat helps to evaporates the ethanol in the previous reagent. That was what my colleague told me, though I am not too sure how the evaporation of the ethanol helps.

#19 tuhin_genes

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Posted 10 March 2009 - 06:19 PM

Hi,

I seem to be getting a low yield (15ng/ul) when trying to purify my DNA from 1% agarose gels with the Qiagen PCR Purification kit. I have two vectors - in two seperate reactions 1 has been cut with AflII & the other with Sal I & ran on a gel. Got strong band for each cut vector on their respective gels but when i cut out & purify the yield seems to be really low. Any suggestions?

hello
when you are gel isolating the bands, for how long you were exposing the vector DNA on the gel doc? It might so happen UV nicks the DNA when exposed so long , moreover you should try the preparative UV which has got longer wavelength than the shorter ones.
It may also happen that the Ultra salt may be too old ( NaI) to dissolve the gel completely and the silica beads may be old to bind to the DNA. try to vortex the silica mixture with the DNA so that the Brownian motion continues and when you precipitate by centrifuging , you get a lots of silica attached to the DNA.
cheers
tuhin k guha

#20 Kami23

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Posted 12 March 2009 - 09:22 AM

Hi,

I seem to be getting a low yield (15ng/ul) when trying to purify my DNA from 1% agarose gels with the Qiagen PCR Purification kit. I have two vectors - in two seperate reactions 1 has been cut with AflII & the other with Sal I & ran on a gel. Got strong band for each cut vector on their respective gels but when i cut out & purify the yield seems to be really low. Any suggestions?

hello
when you are gel isolating the bands, for how long you were exposing the vector DNA on the gel doc? It might so happen UV nicks the DNA when exposed so long , moreover you should try the preparative UV which has got longer wavelength than the shorter ones.
It may also happen that the Ultra salt may be too old ( NaI) to dissolve the gel completely and the silica beads may be old to bind to the DNA. try to vortex the silica mixture with the DNA so that the Brownian motion continues and when you precipitate by centrifuging , you get a lots of silica attached to the DNA.
cheers


Hey,

I have always hated the column extraction kit from Quiagen because the yeild is always so low! but they do a kit which uses a resin called QiaxII which i prefer and has always been ok for me... its a bit more fiddly but well worth it!

#21 Nrelo

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Posted 12 March 2009 - 05:35 PM

Hi,

I seem to be getting a low yield (15ng/ul) when trying to purify my DNA from 1% agarose gels with the Qiagen PCR Purification kit. I have two vectors - in two seperate reactions 1 has been cut with AflII & the other with Sal I & ran on a gel. Got strong band for each cut vector on their respective gels but when i cut out & purify the yield seems to be really low. Any suggestions?

hello
when you are gel isolating the bands, for how long you were exposing the vector DNA on the gel doc? It might so happen UV nicks the DNA when exposed so long , moreover you should try the preparative UV which has got longer wavelength than the shorter ones.
It may also happen that the Ultra salt may be too old ( NaI) to dissolve the gel completely and the silica beads may be old to bind to the DNA. try to vortex the silica mixture with the DNA so that the Brownian motion continues and when you precipitate by centrifuging , you get a lots of silica attached to the DNA.
cheers


Hey,

I have always hated the column extraction kit from Quiagen because the yeild is always so low! but they do a kit which uses a resin called QiaxII which i prefer and has always been ok for me... its a bit more fiddly but well worth it!


the one from viogene also depresses me. sometimes i use pcr kit to purify digested vector, no band prep at all, the yield is much better.

#22 jaregi

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Posted 27 October 2009 - 04:56 AM

I can also confirm that both the Qiagen gel extraction kit as well as its gel purification kit fall well short of their recovery claims of 90-95%. To give you some numbers:
  • 40g* in 1g out = 2.5% :)
  • 10g in 0.5g out = 5% :(
(*40g is well above their stated maximum binding capacity per column of 10g.)

I tested 2 batches of Qiagen kit, NaAc, the 1min wait during elution, eluted with EB not to get the bad recovery due to pH. People in my lab tell me though that it worked better before. They also tried the warming and other tricks to no avail.

#23 swanny

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Posted 27 October 2009 - 06:34 PM

My yields have increased since I started using much more solubilising solution than recommended .
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#24 Vini

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Posted 28 October 2009 - 06:57 AM

My yields have increased since I started using much more solubilising solution than recommended .



we hv started using a kit from another company called RBC. works really well

#25 rukiye

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Posted 09 February 2010 - 09:55 AM

Hi,

when you are gel isolating the bands, for how long you were exposing the vector DNA on the gel doc? It might so happen UV nicks the DNA when exposed so long


Hi

How long should DNA be exposed to UV? is 5 min so long?

#26 vojera

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Posted 10 February 2010 - 09:01 AM

Hi,

when you are gel isolating the bands, for how long you were exposing the vector DNA on the gel doc? It might so happen UV nicks the DNA when exposed so long


Hi

How long should DNA be exposed to UV? is 5 min so long?


I would say it's too long. I try not to go above 30 seconds and even that is pushing it. I've never heard a specific time, but just to go as fast as possible.

#27 Parare

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Posted 14 March 2010 - 09:43 AM

I always warm up the elution buffer (EB, water, etc) to 60C before elution and it helps me a great deal with the concentration.


Why would that help?




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