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Old 05-09-2015, 02:36 PM   #1
SDPA_Pet
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Default Illumina Purification Kits

Hi guys,

I am doing illmumina amplicon sequencing and metagenomics sequencing from environmental samples. We just purified the a plate. I measured the DNA concentration before and after purification. I found there is only 2% re-coverage. Total DNA concentration less than 1ng/ul after purification. This is horrible.

What we used is a gel purification kit and so inefficient (slow).

what DNA purification kits do you normally use for the illumina sequencing? for Nextera kits? We just start to do Illumina sequencing. Back to old days, we did 454 sequencing and it never happened like this.

Any efficient and better kits recommend?
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Old 05-09-2015, 04:14 PM   #2
nucacidhunter
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This depends on application and size distribution of fragments and target range. A highly used method is purification beads such as AMPure. It enables one to customise purification with size selection by using various ratios of beads to DNA solution. Gel purification is suitable for applications requiring very narrow size range. All Illumina protocols including Nextera kits includes instruction for purification steps.
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Old 05-09-2015, 04:36 PM   #3
SDPA_Pet
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If the size of my amplicon is around ~ 500kb, which one you think is better? AMpure or gel?

I don't think the amplicon is narrow size range.
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Old 05-10-2015, 01:51 AM   #4
nucacidhunter
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I assume you mean 500bp. If there is no none-specific product larger than 200 bp and the aim is to clean up and remove primer dimers, a 0.65x clean up with bead would be fine. A narrow size range normally is 10-15% variation from the average fragment sizes distribution. For instance, a library fragment size distribution in 450-550 bp would be narrow.
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Old 05-10-2015, 02:04 AM   #5
SDPA_Pet
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Well, yes. My expected amplicon fragment size is about 500bp. The unwanted products(primer dimers) should be less than 200bp. So the difference between them is quite large. I guess I can use AMPure. We have never used AMPure before. We are using gel purification now. It's not very efficient. Before we try AMpure, I want to know some details. If I want to purified 96 samples using the AMPure, how long does it normally take?

Thanks.
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Old 05-10-2015, 03:35 AM   #6
nucacidhunter
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1- Add 0.65x bead to PCR product (65 ul bead to 100 ul reaction) and mix
2- Incubate at room temperature for 5-10 min (longer incubation will increase recovery)
3- Place the plate on magnetic plate and wait for up to 5 minutes until solution clears
4- Aspirate cleared solution from wells and discard
5- Add 180-200 ul 80% ethanol to each well, incubate for 30 sec then aspirate out the ethanol and discard.
6- Repeat ethanol wash one more time
7- Let plate to sit on magnet at RT for 5-10 min or more until beads dry (couple of cracks appear on beads)
6- Elute DNA with a suitable buffer and volume for down stream application to obtain required concentration (normally 20-30 ul). This will include addition of buffer on magnetic stand, mixing buffer with beads off magnet, incubation at RT for 2 min, returning plate on magnet and transferring DNA solution after clearing into a new plate.

This should take less than an hour for 96 samples in a plate. You can find detailed instruction in bead manufacturer web site.
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Old 05-10-2015, 03:49 AM   #7
SDPA_Pet
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Hi Thank you very much.
This seems much faster than gel. One more question, how many times do you normally purify. You know you need to do two "PCR"s for Illumina sequencing. 1>It is kind of like real PCR 2> 2nd time, Add indices. I did the purification after each PCR? total twice for my gel purification

Should I just do once? after the 2nd PCR.
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Old 05-10-2015, 04:23 AM   #8
nucacidhunter
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If you are using two step PCR, then two purifications is required, one after each PCR.
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