Antimicrobial Resistance Screening
- Gregg Randolph
@Amanda Dougherty
Basic Procedure:
Create library pool (7ul template limit per rxn, might need to be done per sample)
Denature library
Hybridize to baits RNA targets
Bind hybridized molecules to magnetic beads
Wash away non-bound library
Elute bound library
Amplify eluted library
Supplies Needed:
NEBNext Ultra II DNA library preparation kitsÂ
Pippin Prep Fragment Size selection (maybe)
Product | Quantity | Unit Cost | Cost for 200 | Cost for 96 | Cost for 24 | Reason |
|---|---|---|---|---|---|---|
TruSeq Nano DNA High Throughput Library Prep Kit (96 samples) | 1 | $2840 | Â | $2840 | Base library prep | |
Nextera XT DNA Library Preparation Kit (96 samples) | 2 | $3435 | $6870 | Â | 0 | Base library prep |
Nextera XT DNA Library Preparation Kit (24 samples) | 1 | $905 | 0 | Â | $905 | Base library prep |
IDT® for Illumina® DNA/RNA UD Indexes Set A, Tagmentation (96 Indexes, 96 Samples) | 1 | $672 | $672 |  | $672 (same kit can be used for the 200 sample run) | multiplexing 1-96 samples |
IDT® for Illumina® DNA/RNA UD Indexes Set B, Tagmentation (96 Indexes, 96 Samples) | 1 | $672 | $672 |  | 0 | multiplexing +1-96 samples |
TruSeq DNA CD Indexes (96 Indexes, 96 Samples)Â Â | 1 | $546 | Â | $546 | Â | multiplexing +1-96 samples |
iSeq 2x150 bp sequencing | 1 | $600 | $600 | $600 | $600 | sequencing |
qPCR standards | 1 | $12 | $12 | $12 | $12 | standard curve |
qPCR plate check | 2 | $0.05 | $0.10 | Â | $0.05 | qc |
bioanalyzer check | 1 | $40 | $40 | $40 | $40 | qc |
Reamplification steps | 192 | $10 | $1920 | $960 | $240 | Â |
Total Estimate | Â | Â | $10786 | $4998 | $2470 | Â |
Questions:
Are John Calder’s kits available?
How many samples? 200 sample multiplex level, smaller proof of concept
Which Illumina Kit is best? (TruSeq or Nextera). Nextera allows for higher multiplexing, I believe.
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