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Old 07-15-2019, 06:18 AM   #1
Cistron
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Question Removal of <110bp primer-dimers without beads?

Hi all,

I was wondering whether anyone has come across a column-based cleanup kit, which would remove large primers (EMP 16S design) and primer-dimers of around 100bp. Or alternatively, protocols to "abuse" silica columns to behave accordingly.

I'm running a course with students, so I am pretty sure the budget won't stretch for beads and magnetic plates. Unfortunately, given my number of students and time-constraint, I cannot always do the right thing and get the PCR optimised properly - I'm happy it works most of the time .

I've also come across a sample recently (raw milk), where all my troubleshooting wouldn't get rid of the pesky primer-dimers. So I can appreciate that this would happen to one of my flock.

Thanks!
Mike
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Old 07-15-2019, 10:09 AM   #2
luc
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The beads should be cheaper than the columns, you do not need to use the m most expensive ones (Ampure). You can get also cheap magnet setups from ebay.

Zymo has columns for size selection, I do not know details and have never tried them:
https://www.zymoresearch.com/product...ncentrator-kit
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Old 07-15-2019, 10:14 AM   #3
teuthbrush
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Have you looked into making your own beads, starting with Seramag Speedbeads? It's very cheap and works pretty well.
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Old 07-15-2019, 11:21 AM   #4
Cistron
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Thanks for your help!

Quote:
Originally Posted by luc View Post
You can get also cheap magnet setups from ebay.
Not sure I can get a purchase order number for that (), but do you have examples?

Quote:
Originally Posted by teuthbrush
Have you looked into making your own beads, starting with Seramag Speedbeads? It's very cheap and works pretty well.
No! That sounds neat. Are there any published protocols around?
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Old 07-15-2019, 12:17 PM   #5
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Quote:
Originally Posted by Cistron View Post
Thanks for your help!

Not sure I can get a purchase order number for that (), but do you have examples?

No! That sounds neat. Are there any published protocols around?
https://ethanomics.files.wordpress.c...apure_v2-2.pdf

OpenWetware also has one that is a bit more in-depth: https://openwetware.org/wiki/SPRI_bead_mix
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Old 07-15-2019, 10:33 PM   #6
luc
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search Ebay for "Magnetic Bead Separation Rack for 96-Well PCR Tube Plate AMPure illumina"
or
"Magnetic beads separator rack for Ampure beads made for 0.3 mL PCR tubes", $29,
or "Magnetic Beads Separator Rack Stand Ampure 1.5 mL Eppendorf Tubes Separation"
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Old 07-15-2019, 11:18 PM   #7
Cistron
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Quote:
Originally Posted by teuthbrush View Post
Ah, super!

Quote:
Originally Posted by luc View Post
search Ebay for "Magnetic Bead Separation Rack for 96-Well PCR Tube Plate AMPure illumina"
or
"Magnetic beads separator rack for Ampure beads made for 0.3 mL PCR tubes", $29,
or "Magnetic Beads Separator Rack Stand Ampure 1.5 mL Eppendorf Tubes Separation"
Thanks a bunch!

Anyone experimented with the DIY versions off Thingiverse?
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