Coligo Check

Owned by Gregg Randolph
Last updated: Sept 23, 2022
1 min read

@Félix Brédoire

Plan:

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 6LVD3 and processed normally otherwise. The base name for this plate was 6GTL2.