Initial Sample Processing - Alfalfa 16S/ITS

Sample Processing for _END & _EPI plates:

1. Vortex and quick spin deep well plates.

2. Use the 50 uL Integra expandable spacing pipette to transfer 35 uL from each well into a transparent PCR plate. Label the plates as shown in this document Initial Sample Processing - Alfalfa 16S/ITS .

3. Coligos added during extraction so do NOT add during sample processing.

4. Seal, vortex, and spin aliquot plate.

5. 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

1. 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.

2. 20uL of each well of each plate was transferred to a transparent PCR plate and 4uL of coligos were added.

3. Plates were then quantified and normalized to 10ng/uL.

II. GBS Plates 5-7 & 9

1. 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.

2. 20uL of each well of each plate was transferred to a transparent PCR plate and 4uL of coligos were added.

3. Plates were then quantified and normalized to 10ng/uL.

III. GBS Plates 10 & 11

1. Volumes in these plates were short so these plates were vacufuged and then resuspended in 30uL of TE before use.

2. 20uL of each well of each plate was transferred to a transparent PCR plate and 4uL of coligos were added.

3. Plates were then quantified and normalized to 10ng/uL.