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:
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MagBead Cleanup (16S Only):
Some plates were cleaned up using the Nimbus platform protocol “AxyPrep MagBead PCR1 No MM”
Manually, it was done:
Equilibrate Beads to room Temperature
Add 24 ul of MagBeads to each well and 15 ul of replicate to same well of replicate
Pipette mix up and down 10 times.
Incubate at RT for 5 minutes
Secure plate on magnet plate; incubate at RT for 5 minutes (until wells are clear)
Remove 65 ul from each well; keep tips to left or right depending on the column to avoid bead pellet.
Add 100 ul Fresh 80% EtOH to each well. Incubate 30 seconds. Remove 100 ul from each well
Add 100 ul Fresh 80% EtOH to each well. Incubate 30 seconds. Remove 100 ul from each well
Reaspirate from each well to assure maximum EtOH removal
Allow plate to air dry for 7 minutes.
Remove sample plate from magnet plate.
Add 40 ul TE; pipette mix 10+ times. Incubate 2 minutes at RT.
Place sample plate back on magnet for 5 minutes or until all wells are cleared.
Transfer 40 ul to labeled transparent plate (Plate name_MIDs)
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Full result report can be viewed below:
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Pool qPCR results:
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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.