Wagner Trout - RePCR Pool3 of First 12 plates (Trout1)

Should we not prep this MM ahead of time?

PCR Amplification 

PCR1:

Add 16 ul of PCR1 MM to each well of four new hard shell PCR plates with 8 channel

Add 4uL of template from pooled plates with Benchsmart

Seal, Vortex, Spin down, and then run on Thermocycler program: GBS1:

*pause step

Extra PCR

Add 2.125 ul of the MM directly to the previous PCR

Then continue with the last four unlisted steps for GBS1 from above:

Pippin Prep Size Select (300-365 bp select):

Run Final Product on qPCR for check

Result from qPCR check:

Seq Run:

Dilute to 1 nM based off qPCR results. qPCR results are in pM, but 1:1000 dilution used. The results are effectively in nM for pool.

• 1000/Results = ul of Pool to Add

100/1 = 5.5 uL of Pool to Add

• 100 - uL of Pool to Add = ul of “10 mM Tris 8.5” to Add

100- 5.5 = 94.5uL of 10mM Tris 8.5

Dilute 1 nM full pool to loading concentration of 100 pM:

• Add 10 ul 1 nM Pool to 90 ul “10 mM Tris 8.5”

• Remove iSeq 100 i1 Flow Cell from refrigerator 5’s crisper drawer and open white foil pack and allow to equilibrate to RT for 10-15 minutes.

• Open “iSeq 100 i1 Reagent Cartridge v2”. Turn on iSeq100

• Click on “Sequence”. Watch Video. Do what video tells you to do. Follow on screen instructions until run starts.