EcoR1 oligo adapter Stock Dilutions

NOTES: It was communicated that stock plates were 20uL at 100uM so working plate 4 was made based on those numbers. Later it was discovered in Erin’s notes that the stock plates were actually 15uL at 100uM. After confirming what volume was left over after Erin’s creation of the current working plates (minus working plate 4), it seems that Erin’s numbers were correct. Due to this, adjustments have been made below to the instructions on how to create 25uM barcode stock plates. Further adjustments will be necessary to ensure barcode stock 7 and barcode stock 8 are indeed 25uM. Working plate 4 may also need to be recreated since it is assumed to be 0.7uM and not 1uM.

EcoR1 Working Plate 4 Creation (completed in March 2021 for Alfalfa project)*:

SpeedVac Stock Plate 7 and Stock Plate 8 to dry oligos

Resuspend with 20 ul H2O and 60 ul std TE to avoid over saturating with Tris and EDTA to create 25 uM stocks.

Combine 4 ul from each plate in a well to well fashion with 92 ul of TE to create a 1 uM working plate.

Label it “GBS Working Stock Plate 4” Seal, Vortex, and Spin down resulting plate.

Heat to 95C for 5 minutes and allow to slowly cool to RT

Instructions to make 25uM barcode stock plates:

SpeedVac Stock Plates to dry oligos

Resuspend with 14ul H2O and 42ul std TE to avoid over saturating with Tris and EDTA to create 25 uM stocks.

Instructions to make 1uM working plates:

Combine 4 ul from each plate in a well to well fashion with 92 ul of TE to create a 1 uM working plate.

Label it “EcoR1 Working Plate #” Seal, Vortex, and Spin down resulting plate.

Heat to 95C for 5 minutes and allow to slowly cool to RT