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Sample Processing for _END & _EPI plates:
Vortex and quick spin deep well plates.
Use the 50 ml uL Integra expandable spacing pipette to transfer 30 ul 35 uL from each well into a transparent PCR plate. Label the plate as shown here.
If it is only a few shy of a full plate, add 30 ul TE to fill out the last column and treat as samples. (ie. Abby’s second plate)
Vortex and spin control oligo pool (16S and ITS coligo 0.01 pg/ul each AND 16S SG_GR and ITS SG_GR 0.03 pg/ul each. This should be in the left hand refrigerator. (MIGHT NEED TO DELETE THIS STEP IF ALL EXTRACTIONS HAD COLIGOS ADDED BEFORE EXTRACTION.)
Add 6 ul control oligo plate to the sample aliquot plate in a well to well fashion (A1 to A1, B1 to B1, . . .) This can be done using an 8 channel or the 96 channel. If you use the 8 channel, you can reuse the dome caps on the control plate as long as you avoid contamination. Return control plate. Label side opposite numbers of aliquot plate with an asterisk.(MIGHT NEED TO DELETE THIS STEP IF ALL EXTRACTIONS HAD COLIGOS ADDED BEFORE EXTRACTION.)plates as shown in this document Initial Sample Processing - Alfalfa 16S/ITS .
Coligos added during extraction so do NOT add during sample processing.
Seal, vortex, and spin aliquot plate.
Make a new folder under DNA Quantification labeled “Alfalfa Buerkle Lab 16S/ITS”. Check concentration on Synergy HTX and save file on petalibrary as plate name. Add a circle around the asterisk when concentrations have been measured. Normalize plates on the Hamilton Nimbus to 10ng/uL.
Sample Processing for _GBS Plates:
I. GBS Plates 1-4 & 8
GBS plates 1-4 and 8 were normalized for GBS to 100ng/uL (or less) in 30uL. Due to this, these plates had 5uL of TE added to each well to make the quantity for quantification and normalization sufficient. The plates were then vortexed and quick spun.
20uL of each well of each plate was transferred to a transparent PCR plate and 4uL of coligos were added.
Plates were then quantified and normalized to 10ng/uL.
II. GBS Plates 5-7 & 9
Certain wells on these plates were diluted to fit within the parameters required for GBS, due to this the volumes in this plate were inconsistent. Wells that did not have enough volume 16S/ITS were brought up with 5-10uL of TE. Plates were then vortexed and spun down.
20uL of each well of each plate was transferred to a transparent PCR plate and 4uL of coligos were added.
Plates were then quantified and normalized to 10ng/uL.
III. GBS Plates 10 & 11
Volumes in these plates were short so these plates were vacufuged and then resuspended in 30uL of TE before use.
20uL of each well of each plate was transferred to a transparent PCR plate and 4uL of coligos were added.
Plates were then quantified and normalized to 10ng/uL.