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Low Sample (LS) Protocol
2
Combine Sample Purification Beads and PCR grade water in a tube to create a diluted
bead mixture of 160 µl per 100 µl of end-repaired sample:
• When processing > 6 samples at a time, use a new 15 ml conical tube
• When processing ≤ 6 samples at a time, use a new 1.7 ml microcentrifuge tube
Determine the volumes using the following formulas, which include 15% excess for
multiple samples:
Table 8 Diluted Bead Mixture for a 350 bp Insert Size
Formula
Sample Purification Beads
PCR grade water
# of samples X
109.25 µl
# of samples X
74.75 µl
Table 9 Diluted Bead Mixture for a 550 bp Insert Size
Formula
Sample Purification Beads
PCR grade water
Example
Amount
per 12
samples
# of samples X
92 µl
# of samples X
92 µl
Your
Calculation
1311 µl
897 µl
Example
Amount
per 12
samples
1104 µl
Your
Calculation
1104 µl
3
Vortex the diluted bead mixture for 5 seconds to make sure that the beads are evenly
dispersed.
4
Add 160 µl of the diluted bead mixture to each well of the IMP plate. Set a 200 µl
pipette to 200 µl, and then gently pipette the entire volume up and down 10 times to
mix thoroughly.
NOTE
Aspirate the diluted bead mixture slowly and dispense it slowly due to the viscosity of
the solution. Changes in the volume of the diluted bead mixture affect the insert size of
your library.
NOTE
Vortex the diluted bead mixture frequently. Illumina recommends the following:
• If using a single channel pipette, vortex the mixture after processing 4 samples
• If using a multichannel pipette, vortex the mixture after processing 4 columns
• If the mixture is in a reagent reservoir, mix with a 1000 µl pipette.
5
Incubate the plate at room temperature for 5 minutes.
6
Place the plate on the magnetic stand for 5 minutes or until the liquid is clear.
7
Set a 200 µl single channel or multichannel pipette to 125 µl. Transfer 125 µl of the
supernatant, which contains the DNA of interest, from each well of the IMP plate to
the corresponding well of the new 0.3 ml PCR plate labeled with the CEP barcode.
Take care not to disturb the beads.
NOTE
Transfer, do not discard, the supernatant. It contains the DNA of interest.
8
18
Repeat step 7. Each CEP plate well now contains a total of 250 µl of DNA of interest.
Part # 15036187 Rev. C