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Three plate experiment for inclusion on Micro NovaSeq 6 or 7. One plate will only use the coligo and ISD as a template; this will double check that all coligos amplify and sequence. One of the regular plates for the prep will be prepped twice for the other two. One will be a standard prep and the second will have a newly prepared coligo/ISD plate used that doubles the coligo concentration, changing the ratio from 1:3 to 2:3 for coligo to ISD.
Control Only Plate:
2 ul of original strength ISD/coligos (plate 7) were added to the standard 1-step PCR master mix for 2 replicates of 16S and ITS. They were all labeled iterations off “6GTL1”.
Extra Coligo Plate:
A coligo/ISD plate was created with double the coligo concentration:
- Dilute 30 ng/ul ISD to 10 ng/ul
- 200 ul ISD
- 400 ul TE
- Combine 10ng/ul ISD and 10 ng/ul coligos to get 30pg/ul ISD and 20 pg/ul coligo plate
- 995 ul TE
- 3 ul ISD
- 2 ul coligos
- Dilute new plate to get 0.3 pg/ul ISD and 0.2 pg/ul coligo
- 495 ul TE
- 5 ul coligo/ISD
- Dilute new plate to get 0.03 pg/ul ISD and 0.02 pg/ul coligo
- 450 ul TE
- 50 ul coligo/ISD
2 ul of this plate was then added to 20 ul from 6LVD? and processed normally otherwise. The base name for this plate was 6GTL2.