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Old 09-02-2015, 01:26 PM   #1
HannahOish
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Default Optimizing DNA Extraction from Dried Insects

Hi all,
I am attempting to get enough (and good enough) DNA extracted from dried adult butterflies to do sequence capture. I've been told I need at least 1 microgram of DNA for guaranteed results, which is a bar I have not yet met (0.952 micrograms is the most I've gotten, to date).

I have been mechanically grinding a single leg per specimen, and then using overnight incubation with proteinase K, followed by phenol chloroform extraction (the commercial Omniprep kit), according to the standard procedure (except that I'm using Glycoblue instead of the glycogen that comes with the kit).

Does anyone have recommendations for tweaks this procedure, or other procedures that may increase yeild?

Otherwise, when do you say enough's enough (for quality and quantity) and send it off and hope for the best?

Thanks for your thoughts.
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Old 09-02-2015, 03:12 PM   #2
kerplunk412
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Maybe this is a stupid question, but could you just process a couple more legs and combine the samples with ethanol precipitation later?
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Old 09-02-2015, 06:32 PM   #3
JamesH
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Sometimes soaking dried insects in TE overnight before the extraction can help with recovery
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Old 09-03-2015, 08:19 AM   #4
HannahOish
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Not a stupid questions at all, kerplunk412. I'm trying to find the sweet spot where I can still get enough DNA to do NGS, without destroying half the specimen--these are museum specimens, and some of the species are quite rare and not easily replaced.

Good suggestion, JamesH.
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Old 09-03-2015, 08:27 AM   #5
lre1234
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What about taking the DNA that you have gotten and try doing MALBAC or some other whole genome amplification (not sure how well this would work)
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Old 09-03-2015, 12:57 PM   #6
HannahOish
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We have a kit for WGA on order. While I'm waiting, I'm trying to collect other ideas in case that doesn't work well. I'm concerned that since the DNA in some cases is already rather degraded, I'll just end up amplifying junk. Worth a shot, though.
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Old 10-10-2015, 07:56 AM   #7
han526
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Have you found a solution yet? What is your PK digestion conditions?

Quote:
Originally Posted by HannahOish View Post
We have a kit for WGA on order. While I'm waiting, I'm trying to collect other ideas in case that doesn't work well. I'm concerned that since the DNA in some cases is already rather degraded, I'll just end up amplifying junk. Worth a shot, though.
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Old 11-11-2015, 05:48 AM   #8
Lucigen_NGS_Service
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HannahOish,

May I ask what the range of yields you have found so far? I ask because if your goal is to do WGS for rare specimens, where sample preservation is at a premium, you may already have enough material to construct good, high-complexity libraries without amplification.
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