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Old 05-07-2019, 12:01 PM   #1
pmiguel
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Location: Purdue University, West Lafayette, Indiana

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Default Best method to add custom primer to MiSeq Illumina sequencing primers?

What pipette is recommended (or have you seen used) to pipette custom primer into the Illumina sequencing primer wells of a MiSeq? (See here :
https://support.illumina.com/bulleti...-primers-.html

if it isn't clear what I mean.)

Currently we use some 1ml glass serological pipettes that were purchased over a decade ago. They actually work, but only poorly. We pipet out the liquid in Cartridge Positions 12 and/or 13 and/or 14 into eppendorf tubes. Mix in the (concentrated) custom primers to 0.5uM and pipet the mixture back into the position from which it came.
But if there existed a long pipette tip that fit on the end of a normal pipetter (p1000), that would be preferable.

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Old 05-09-2019, 04:02 AM   #2
kmcarr
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Quote:
Originally Posted by pmiguel View Post
What pipette is recommended (or have you seen used) to pipette custom primer into the Illumina sequencing primer wells of a MiSeq? (See here :
https://support.illumina.com/bulleti...-primers-.html

if it isn't clear what I mean.)

Currently we use some 1ml glass serological pipettes that were purchased over a decade ago. They actually work, but only poorly. We pipet out the liquid in Cartridge Positions 12 and/or 13 and/or 14 into eppendorf tubes. Mix in the (concentrated) custom primers to 0.5uM and pipet the mixture back into the position from which it came.
But if there existed a long pipette tip that fit on the end of a normal pipetter (p1000), that would be preferable.

--
Phillip
Phillip,

Our lab does essentially the same as you, remove solution from cartridge, mix in Eppendorf, return to cartridge, but they use disposable Pasteur pipets to suck the sample out of and return it to the cartridge well. Almost every MiSeq run we do uses custom primers (amplicon metagenomics) and I believe the folks in the lab are happy with this solution.
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Old 05-24-2019, 01:18 PM   #3
thermophile
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I use a long p200 (https://www.usascientific.com/200ul-...uated-tip.aspx). Pipet up ~150ul of primer in well, mix with aloquated primer in epitube, add back to well, pipet up and down to mix.
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