Take out 6 tubes of dNTPs and 9 tubes of 5x Buffer to thaw.
Get new 50 ml tube from pre-PCR side. Volumetric dispense 30 ml from Water Filer system. Remove 275 ul to reach 29725 ul. Label tube.
Take out 5 tubes of Taq when ready to assemble.
ul/rxn | Reagent | # of rxns | ul needed |
|---|---|---|---|
3 | 5X Kapa HiFi Buffer | 4100 | 12300 |
0.45 | 10M dNTPs | 4100 | 1845 |
0.3 | Kapa HiFi HotStart DNA Pol | 4100 | 1230 |
7.25 | HPLC H2O | 4100 | 29725 |
11 | Total Volume | 4100 | 45100 |
Aliquot 4,490 ul into 10 5ml tubes and label each A_mmddyy_AmpliconStdMM_#
Aliquot 11 ul into each well of an 8 pcr-tube strip. Add 4 ul IlluminaTest primers to 2. Add 4 ul 16STest primers to 1; add 2 ul 16STest primers to the next and 2 ul TE. Add 4 ul ITSTest primers to 1; add 2 ul ITSTest primers the next and 2 ul TE., Add 2 ul 16STest primers and 2 ul MC to the last 2. Add Test date to TE source.
Cap, Vortex, and spin down. Run on thermocycler program GSAF36:
Temp C | Cycles | Time |
|---|---|---|
95* | 1X | 3:00* |
98 | 36X | 0:30 |
62 | 36X | 0:30 |
72 | 36X | 0:30 |
72 | 1X | 5:00 |
4 | 1X | 0:00 |
No cleanup needed. Run resultant rxns on a D1000 High Sensitivity Tape.
Remove reagents and tape out of fridge for 30 minutes
Vortex and spin down Buffer and Ladder
Add 2 ul High Sensitivity Buffer to 9 wells of 2 empty Agilent Optical Tube Strips
Add 2 ul Ladder to the first well of one.
Add 2 ul sample to each well you added Buffer.
Apply caps, Label tops, and quick spin down.
Transport to INBRE.
Open TapeStation Software
Add Tape to slot with QR code in the back right
Vortex tube strips for 1 minute at 2000 rpm
Assign sample names in software
Spin tubes strips down for 1 minute
Add samples info instrument with ladder in A1 (back left)
After run completion, Create Report and add to report subpage
email or use usb to transfer data
After use, save any remaining MM in 2 ml tube. Transfer appropriate label for post sequencing check if necessary.