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Old 05-26-2009, 04:26 PM   #1
greigite
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Question gel extraction protocols

Hi,
I'm hoping to collect some information on how you all handle the gel extraction step in the Illumina sample prep protocol. I have had some issues getting a decent yield of DNA out of 2% TAE agarose gels with the Qiagen QiaexII kit. What kits do you find work particularly well? Any tips for sterile gel prep, visualization, and band excision? Does EtBr cause downstream problems? Does anyone use the new(ish) high voltage electrophoresis buffers (Na-borate/Li-borate/Li-acetate) and/or visual stains (crystal violet/methylene blue)?
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Old 05-27-2009, 03:08 PM   #2
ScottC
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I do it in a pretty basic/standard sort of way... run a 2% TAE gel (Fermentas TopVision LE GQ low EEO agarose, 'home made' TAE) at 100V for an hour alongside 100bp ladder from NEB. For visualisation I use SybrSafe stain (Invitrogen) and a blue LED illuminator (Invitrogen) on which I cut the bands using sterile scalpel blades and transfer to 1.5mL eppendorf tubes. I use the Qiagen Qiaquick gel extraction kit as per the manufacturers instructions except that I don't do the 50oC incubation step to melt the gel. I do it for 10 minutes at room temperature. It usually works well for us, but that's having started the procedure with 1-5 ug of genomic DNA.

Cheers,

Scott.
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Old 06-08-2009, 04:18 PM   #3
greigite
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Default UV damage?

Thanks for your protocol, Scott. I wonder if anyone has looked the effects of UV damage on base representation in downstream sequencing? I might expect there to be some effect at least...
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Old 08-17-2009, 02:34 AM   #4
Exeplex
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Default Gel extraction

Scott's tip about not heating during gel dissolution is a good one - see the paper by Quail et al. (Nature Methods, December 2008) for more details - though the time needed for complete solubilization may be >10 minutes depending on ambient temperature and the size of gel slice.

To maximise DNA recovery from the column, I'd also recommend heating the elution buffer to 65 degrees C, then leaving it on the column for at least 3 (but not more than 5) minutes before spinning - doing this generally gives >95% yield from the column in a single 30ul elution.

Quote:
Originally Posted by ScottC View Post
I do it in a pretty basic/standard sort of way... run a 2% TAE gel (Fermentas TopVision LE GQ low EEO agarose, 'home made' TAE) at 100V for an hour alongside 100bp ladder from NEB. For visualisation I use SybrSafe stain (Invitrogen) and a blue LED illuminator (Invitrogen) on which I cut the bands using sterile scalpel blades and transfer to 1.5mL eppendorf tubes. I use the Qiagen Qiaquick gel extraction kit as per the manufacturers instructions except that I don't do the 50oC incubation step to melt the gel. I do it for 10 minutes at room temperature. It usually works well for us, but that's having started the procedure with 1-5 ug of genomic DNA.

Cheers,

Scott.
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Old 11-22-2009, 09:23 PM   #5
treebeard
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All - our group was also concerned by the comment in Quail et al (Nature Methods 2008) relating to the biased recovery of DNA fragments, possibly due to the Qiagen process.

To get around this, we've been fractionating our Illumina libraries on 2.5% low-melting point agarose (Promega) that contain GelRed (Biotium). We isolate libraries in the same way noted by ScottC (above), except that we do not use Qiagen columns for subsequent clean-up. Rather, we simply melt the gel slice and use the melted agarose/DNA mixture in the subsequent PCR enrichment step (typically 2 - 4 ul in a 50 ul PCR reaction). This is a very fast and very reliable alternative - we've used it in the construction of 100+ libraries and have had few failures.

If you decide to try this, be sure to modify the TAE buffer so that EDTA is at 0.1X the normal concentration (carry-over of EDTA into the PCR enrichment step inhibits the reaction).
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Old 11-23-2009, 09:00 AM   #6
Marta
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I use 2% agarose in TBE with GelRed followed by fragment extraction using NucleoTrap from Macherey-Nagel (or Clontech). I did not see any problems with 50C incubation for agarose melting step in NucleoTrap lysis buffer.
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Old 11-24-2009, 07:22 AM   #7
greigite
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Recently I've had some success using the visible stain nile blue to stain my gels prior to extraction to avoid UV damage. I've found that 2 micrograms per ml in the gel and the running buffer gives good visibility, which can be improved by destaining if necessary. You do need to use more ladder- 25 to 30 ul works well for me. You can see the stain using a white light plate. I make a 1000x stock of nile blue in methanol.
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Old 11-24-2009, 07:23 AM   #8
greigite
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have you tried this with sodium borate or lithum acetate buffers? That would eliminate the EDTA problem.

Quote:
Originally Posted by treebeard View Post
All - our group was also concerned by the comment in Quail et al (Nature Methods 2008) relating to the biased recovery of DNA fragments, possibly due to the Qiagen process.

To get around this, we've been fractionating our Illumina libraries on 2.5% low-melting point agarose (Promega) that contain GelRed (Biotium). We isolate libraries in the same way noted by ScottC (above), except that we do not use Qiagen columns for subsequent clean-up. Rather, we simply melt the gel slice and use the melted agarose/DNA mixture in the subsequent PCR enrichment step (typically 2 - 4 ul in a 50 ul PCR reaction). This is a very fast and very reliable alternative - we've used it in the construction of 100+ libraries and have had few failures.

If you decide to try this, be sure to modify the TAE buffer so that EDTA is at 0.1X the normal concentration (carry-over of EDTA into the PCR enrichment step inhibits the reaction).
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