RM_JAN22 is being repeated starting at PCR to increase the diversity of MIDs. Since we used two consecutive 16S MID plates for the first PCR, we suspect that this is causing the clusters to be too similar to properly pass through the flow cell. To fix this issue, the new PCR set up only used one MID plate (16S0H616S0H5) and was set up as follows:
...
Full result report can be viewed below:
| View file | ||
|---|---|---|
|
Pool qPCR results:
| View file | ||
|---|---|---|
|
iSeq 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
1000/32.25 = 31uL of Pool to Add
1000 - uL of Pool to Add = ul of “10 mM Tris 8.5” to Add
1000- 31 = 969uL of 10mM Tris 8.5
*1nM pool will be combined with 1nM pool for the low read 2.5 experiment. Directions for iSeq prep found here.