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Old 11-21-2011, 08:18 AM   #1
shawpa
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Default library gel purification with SYBR gold problems

We are using the Illumina truseq DNA adapter prep kit. We are now doing whole genome and went to using the SYBR gold as illumina suggested. I had used SYBR safe and gotten low yield so they suggested SYBR gold as per protocol. We have done it twice and our ladders and/or samples are not running properly. Our 100bp ladder just looks like a big smear. These were about 2% agarose TAE gels. The ladder is from bioexpress. Is there something about SYBR gold that would be causing this? Is there some change we need to implement? Just to clarify, we add 15ul SYBR gold to about 120ml agarose pre gel-electrophoresis.
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Old 11-21-2011, 10:54 AM   #2
pmiguel
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We never tried this--the instructions for SYBR-gold imply that it must be used only as a post-stain. I was surprised to see Illumina recommending that it be used during the run.

Also, I don't see how using SYBR-gold instead of SYBR-safe would increase your yield. I had only seen SYBR-gold suggested as a method to avoid the forked-adapter migration artifacts that one sometimes sees. But I would much prefer ETHANol's doing a few cycles of PCR prior to running the gel method to swapping out my running dye.

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Old 11-22-2011, 04:59 AM   #3
bbeitzel
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You may need to cut back on the amount of ladder that you are loading. I have seen that when using very sensitive dyes (GelStar, GelRed, most of the Sybr stains) you need to cut back on the amount of ladder that you use by 5-10x. If you use the same amount as you would for an EtBr gel, you end up with a big smear.

I agree with pmiguel, too, as I thought Sybr-Gold is supposed to be used for post staining only.
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Old 01-16-2012, 12:25 PM   #4
Gina_P
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I have used SYBR gold with great success, mixed into the gel as Illumina suggests. I used Benchtop ladder, which was also suggested in the manual, which does resolve quite clearly. It could be that the Bioexpress ladder is created in such a way that it does not resolve well with SYBR gold staining (maybe it has overhangs) - try looking up that information or maybe using less ladder as the others suggest.
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