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The TRNL pool via standard size estimation returned a mean of ? nM. This should be adjusted for the difference between the standards' fragment sizes and the expected product size (452 vs 220). ?x9.41x(452/220) = ? 19.33 nM
16S is close enough to the standards' fragment size that the standard estimation can be used. 16S mean is 46.34 nM.
iSeq Sequencing:
Dilute TRNL Pool 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/
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19.33 =
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52uL of Pool to Add
1000 - uL ul of Pool to Add = ul of “10 mM Tris 8.5” to Add
1000-
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52 =
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948 uL of 10mM Tris 8.5 to Add
Pool 16S Pool 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/
...
46.34 = 22 uL of Pool to Add
1000 - uL of Pool to Add = ul of “10 mM Tris 8.5” to Add
1000-
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22 =
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978 uL of 10mM Tris 8.5
Combine 100 ul TRNL 1 nM pool to 100 ul 16S 1 nM pool to create a combined 1 nM pool
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