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Gel purification of vector DNA - low yield


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#1 moorele

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Posted 23 February 2009 - 08:34 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?

Edited by moorele, 23 February 2009 - 08:35 AM.


#2 microgirl

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Posted 23 February 2009 - 08:49 AM

The yields from Qiagen gels are always low in our lab's experience. You could try a different kit - someone once suggested Zymo to me.

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?



#3 moorele

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Posted 23 February 2009 - 08:54 AM

I've never had a problem with the yield before. I usually get about 2ug/30ul so dont think its down to the kit. Plus others in my lab have been using it recently and havent had a problem with their yields.

I eluted in dH20 which a colleague has said usually results in a lower yield but not as low as i've gotten. I'm going to try eluting with Buffer EB diluted 1 in 5 with dH20 & see if that increases the yield.

#4 perneseblue

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Posted 23 February 2009 - 09:15 AM

might you have tried passing the QC buffer (yellow) which has the gel dissolve in through the column several times? I pass the solution 3 times. It gives the DNA more opportunity to bind to the column.

Also adding enough QC buffer to the gel is important. Too much is okay. Too little and the DNA doesn't bind to the silicon column as well.

Adding isopropanol, if you haven't already.

Have you tried eluting the DNA with EB buffer warmed to 68C?
May your PCR products be long, your protocols short and your boss on holiday

#5 moorele

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Posted 23 February 2009 - 09:18 AM

might you have tried passing the QC buffer (yellow) which has the gel dissolve in through the column several times? I pass the solution 3 times. It gives the DNA more opportunity to bind to the column.

Also adding enough QC buffer to the gel is important. Too much is okay. Too little and the DNA doesn't bind to the silicon column as well.

Adding isopropanol, if you haven't already.

Have you tried eluting the DNA with EB buffer warmed to 68C?



I've added isopropanol but havent tried passive the QC buffer through the column a few times! I'll give that a shot. I will also try warming the EB buffer! Thanks for those suggestions. I'll let you know if i have any success!

#6 perneseblue

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Posted 23 February 2009 - 09:33 AM

I've added isopropanol but havent tried passive the QC buffer through the column a few times! I'll give that a shot. I will also try warming the EB buffer! Thanks for those suggestions. I'll let you know if i have any success!


Oh, another trick is to elute the column twice. Let say the EB volume you are using is 50ul. Break the volume of the EB buffer into two. Elute the column first with 20ul. Then a second time with 30ul. This does improve the amount of DNA eluted from the column.
May your PCR products be long, your protocols short and your boss on holiday

#7 jiajia1987

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Posted 24 February 2009 - 05:32 PM

I've added isopropanol but havent tried passive the QC buffer through the column a few times! I'll give that a shot. I will also try warming the EB buffer! Thanks for those suggestions. I'll let you know if i have any success!


Oh, another trick is to elute the column twice. Let say the EB volume you are using is 50ul. Break the volume of the EB buffer into two. Elute the column first with 20ul. Then a second time with 30ul. This does improve the amount of DNA eluted from the column.


Or alternatively, u can elute in 50ul first, stand for 2 mins instead of the usual 1 min, and spin it down. then take the eluate and place it on the exact same spin column again and stand for 1 min and spin it down. u get better recovery this way.

#8 scolix

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Posted 28 February 2009 - 08:50 AM

Which kit do you use? If Qiagen, try adding the additional Na-acetate.

Or try invitrogen's kit, we got better yield with it.

#9 NemomeN

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Posted 01 March 2009 - 07:30 PM

Also, elution buffers need to have a pH of at least 8. ddH20 (surprisingly) is at pH of between 5 and 6.5...and in my experience, this has killed the yields of the column. The EB buffer from Qiagen is actually just Tris pH 8.0 (I think it's 10mM but not positive). If you are using water, try raising the pH to 8

#10 planktonica

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Posted 04 March 2009 - 01:13 PM

I've been getting low yields too using the gel extraction kit from Qiagen.
I add 3M Na-acetate to the sample: didn't work.
I passed QG buffer twice: it washed away my DNA...

I don't know what to do... ;)

#11 perneseblue

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Posted 04 March 2009 - 01:19 PM

I've been getting low yields too using the gel extraction kit from Qiagen.
I add 3M Na-acetate to the sample: didn't work.
I passed QG buffer twice: it washed away my DNA...

I don't know what to do... ;)


did you add enough QG buffer to your gel slab? It is the QG buffer that causes the DNA to bind to the column
May your PCR products be long, your protocols short and your boss on holiday

#12 planktonica

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Posted 04 March 2009 - 01:28 PM

I've been getting low yields too using the gel extraction kit from Qiagen.
I add 3M Na-acetate to the sample: didn't work.
I passed QG buffer twice: it washed away my DNA...

I don't know what to do... ;)


did you add enough QG buffer to your gel slab? It is the QG buffer that causes the DNA to bind to the column


I add 5 times as much QG as my sample volume, as directed in the protocol. I really get low vector concentrations (around 10% recovery) and I am not sure if this is giving me problems with my ligation.

#13 rkay447

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Posted 04 March 2009 - 02:59 PM

The instructions in the Qiagen gel extraction kit say to add 3X the QG buffer as the sample volume. It is the pcr purification kit that requires a 5X buffer addition. Most likely the buffer and isopropanol concentrations are way off. However, you only need a very small amount of vector for ligations so even if you have a bad yield, you should still have enough to get a ligation to work. I typically use 25ng vector in a ligation but have used as little as 10ng with success.

#14 planktonica

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Posted 05 March 2009 - 09:14 AM

My PI told me to use the PCR purification protocol with the gel extraction because we are not actually extracting from gel. But I will try to use 3X instead and see what I get.

Thanks!

#15 swanny

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Posted 05 March 2009 - 03:39 PM

OK, really dumb question, but in which stages do you add the isopropanol and the Na acetate?
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