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In the quest to determine the source of contamination, this test was done to explore MasterMix as the culprit. Medicago was still highly prevalent in the reads. Mastermix was unlikely the sole culprit if a culprit at all. Further inquiries are on another page. |
Master Mixes used with links to QC pages
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Plate Name | Average Result (nanomoles) |
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Full qPCR results below:
Sequencing Test:
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
100/8.4 = 12uL of Pool to Add
100 - uL of Pool to Add = ul of “10 mM Tris 8.5” to Add
100- 12 = 82 uL of 10mM Tris 8.5
Dilute 1 nM full pool to loading concentration of 60 pM:
Add 6 ul 1 nM Pool to 82 ul “10 mM Tris 8.5” and 12 ul 50 pM PhiX
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.
This run failed without detecting any clusters. So, we are repeating it.
Redone Sequencing Test:
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
100/10 = 10uL of Pool to Add
100 - uL of Pool to Add = ul of “10 mM Tris 8.5” to Add
100- 10 = 90 uL of 10mM Tris 8.5
Dilute 1 nM full pool to loading concentration of 60 pM:
Add 6 ul 1 nM Pool to 82 ul “10 mM Tris 8.5” and 12 ul 50 pM PhiX
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.