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Old 05-23-2019, 03:59 PM   #1
SDPA_Pet
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Default NGS clean up kit

Hello, I am want to purify some PCR amplicons (16S rRNA) for Illumina sequencing.

I have many samples and I did my PCR in 96-well plates. I was told the best way to do it is using magnetic beads methods. I order a 96 rxn kits. I thought I could purify 96 sample once. However, I have to transfer sample to a 1.5mL microcentrifudge tube and purify it one by one.

I am looking for a kit can purify 96-well plates once. It's better using megnetic beads methods. I am wondering if anyone can tell me which kits that I should buy. Can you send me the company name, lot number or website link

Thanks,
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Old 05-23-2019, 06:30 PM   #2
luc
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You will need a magnetic bead separation plate to use the beads for cleanups in 96-well PCR plates.

Zymo has DNA clean & concentrate kits in plate format (silica columns). You will require a suitable centrifuge.
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Old 05-24-2019, 09:57 AM   #3
AvantS
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We use AMPure beads at 1.2X and a 96-well bead extractor protocol for our PCR purification. This lets us purify all 96 wells with sample at once and multiple plates in less than an hour.

You would need to multi-channel transfer your samples to a U-bottom plate, which is better than individual 1.5mL tubes imo.
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Old 05-26-2019, 06:58 PM   #4
SDPA_Pet
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Quote:
Originally Posted by AvantS View Post
We use AMPure beads at 1.2X and a 96-well bead extractor protocol for our PCR purification. This lets us purify all 96 wells with sample at once and multiple plates in less than an hour.

You would need to multi-channel transfer your samples to a U-bottom plate, which is better than individual 1.5mL tubes imo.

Hello AvantS,

Thank you very much. Can you give me the details (website link) of AMpure beads 1.2X. I am not sure what is 1.2 X? I heard Ampure beads XP. Are they same thing?

Thanks.
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Old 05-26-2019, 08:42 PM   #5
AvantS
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This is the product we use.

The 1.2X is volume ratio of bead to sample. For example, if I wanted to purify 100uL of 300bp target DNA by removing <100bp primer dimer, I would at 120uL of beads to the sample. The ratio chosen depends on the length DNA you want to keep.


The image below helps illustrate what I mean.

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Old 05-27-2019, 07:41 PM   #6
SDPA_Pet
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Quote:
Originally Posted by AvantS View Post
This is the product we use.

The 1.2X is volume ratio of bead to sample. For example, if I wanted to purify 100uL of 300bp target DNA by removing <100bp primer dimer, I would at 120uL of beads to the sample. The ratio chosen depends on the length DNA you want to keep.


The image below helps illustrate what I mean.


Hi Thanks for you information. I am using a different brand called Geneaid. However, the product comes with very simple protocol. It only says that I can clean up the DNA from 100bp to 10kb. It only tell me every 100uL of PCR product should add 10uL beads, XXX buffer etc.

I don't have any gel picture like this. Is there any equation to calculate how much beads should be added? My primers are about 120bp and Amplicon is about 300bp.
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Old 05-27-2019, 09:17 PM   #7
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Quote:
Originally Posted by SDPA_Pet View Post
Hi Thanks for you information. I am using a different brand called Geneaid. However, the product comes with very simple protocol. It only says that I can clean up the DNA from 100bp to 10kb. It only tell me every 100uL of PCR product should add 10uL beads, XXX buffer etc.

I don't have any gel picture like this. Is there any equation to calculate how much beads should be added? My primers are about 120bp and Amplicon is about 300bp.

I would suggest doing a trial on a ladder with intervals as shown in the image, different brands cut at different ratios so trialling the product in house has always been our best approach.
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