4-15-2022 Samples have been initially pooled and PCRed. They still need to be pooled all together, size selected, qPCRed and bio analyzed.
Check In Samples Against List from Jill Hamilton
Load Submission Data into MISO
Template Plates | ||
---|---|---|
RHIR1 | RHIR2 | RHIR3 |
RHIR4 | RHIR5 | RHIR6 (32 samples) |
Restriction Digestion
(Keep MM and reaction plates on ice)
Set Incubator to 37 C
Add 3 ul Digestion MM to 7 plates using the 8 channel
Reagent | ul/rxn | rxns | ul needed (1.5x) |
10x T4 Buffer | 1.15 | 500 | 862.5 |
5M NaCl | 0.12 | 500 | 90 |
1 mg/ml BSA | 0.6 | 500 | 450 |
H2O | 0.73 | 500 | 547.5 |
MseI (enzyme) | 0.12 | 500 | 90 |
EcoR1 (enzyme) | 0.28 | 500 | 210 |
Total | 3 | 500 | 2,250 |
Add 6 ul template to each plate with Benchsmart. Add 6 ul of TE to Wells B4-H4 of RHIR6. Cover and seal each plate, vortex, centrifuge and incubate at 37C for 8 hours. Followed by 65C in EcoBGC oven for 1 hour.
Adaptor Ligation
Spin down plates and add 1.4 ul Ligation MM to each well with 8 channel.
Reagent | ul/rxn | rxns | ul needed (1.6x) |
---|---|---|---|
MseI oligo | 1 | 500 | 800 |
H2O | 0.112 | 500 | 89.6 |
10x T4 Buffer | 0.1 | 500 | 80 |
5M NaCl | 0.01 | 500 | 8 |
1 mg/ml BSA | 0.05 | 500 | 40 |
T4 DNA ligase | 0.1675 | 500 | 134 |
Total | 1.4 | 500 | 1120 |
Add 1 ul of EcoR1 Adaptors with 8-channel and Track which template plate gets which MID plate.
Template | EcoR1 MID plate |
---|---|
RHIR1 | EcoR1 MID plate 1 |
RHIR2 | EcoR1 MID plate 2 |
RHIR3 | EcoR1 MID plate 3 |
RHIR4 | EcoR1 MID plate 4 |
RHIR5 | EcoR1 MID plate 5 |
RHIR6 | EcoR1 MID plate 6 |
Label reaction plates with MID plate used.
Cover, vortex, and spin plates. Incubate plates at RT for 2 hours on benchtop.
Add 120 ul of low EDTA TE store at 4C for a month or -20C for longer.
PCR Amplification
Create working pools. For Pool A combine ligated plates matching well to well. For pool B combine ligated plates with even plates flipped upside down (H12 in A1). Pool C (or C1 - C4) will be recombining plate 5 by quarter plates and Plate 7 by column. Combine columns 5:1-3, 5:4-6, 5:7-9 and 5:10-12 into columns 1-4 of a new plate. Then into columns 5-8 of the same plate add columns 5:1-3, 5:6-4, 5:7-9 and 5:12-10. Into column 9 of this same plate, add columns 1-4 of RHIR6. Into column 10, add columns 1-4 of RHIR6, but flip columns 2 and 4 upside down.
Pool | Plate1 | Plate2 | Plate3 | Plate4 |
---|---|---|---|---|
A | RHIR1 | RHIR2 | RHIR3 | RHIR4 |
Pool | Plate1 | Plate2 | Plate3 | Plate4 |
---|---|---|---|---|
B | RHIR1 | RHIR2 (FLIPPED) | RHIR3 | RHIR4 (FLIPPED) |
Pool | Plate5 Col 1-3 | Plate5 Col 4-6 | Plate5 Col 7-9 | Plate5 Col 10-12 |
---|---|---|---|---|
C1 | RHIR5 (Col 1-3) | RHIR5 (Col 4-6) | RHIR5 (Col 7-9) | RHIR5 (Col 10-12) |
Pool | Plate5 Col 1-3 | Plate5 Col 6-4 | Plate5 Col 7-9 | Plate5 Col 12-10 |
---|---|---|---|---|
C2 | RHIR5 (Col 1-3) | RHIR5 (Col 6-4) | RHIR5 (Col 7-9) | RHIR5 (Col 12-10) |
Pool | Plate6 Col 1 | Plate5 Col 2 | Plate5 Col 3 | Plate5 Col 4 |
---|---|---|---|---|
C3 | RHIR6 Col1 | RHIR6 Col2 | RHIR6 Col3 | RHIR6 Col4 |
Pool | Plate6 Col 1 | Plate5 Col 2 | Plate5 Col 3 | Plate5 Col 4 |
---|---|---|---|---|
C4 | RHIR6 Col1 | RHIR6 Col2 (FLIPPED) | RHIR6 Col3 | RHIR6 Col4 (FLIPPED) |
PCR1:
Make MM1 in a 15 ml tube:
Reagent | ul/rxn | rxns | ul needed |
---|---|---|---|
H2O | 9.52 | 272 | 3665.2 |
5x iProof buffer | 4 | 272 | 1540 |
10 mM dNTPs | 0.4 | 272 | 154 |
50 mM MgCl2 | 0.4 | 272 | 154 |
5 uM Illumina Primers | 1.33 | 272 | 512 |
iProof TAQ | 0.2 | 272 | 77 |
DMSO | 0.15 | 272 | 57.8 |
total | 16 | 272 | 6160 |
Add 16 ul of PCR1 MM to each well of four new hard shell PCR plates with 8 channel
Add 4uL of template from pooled plates with Benchsmart
Seal, Vortex, Spin down, and then run on Thermocycler program: GBS1:
Temp C | Cycles | Time |
---|---|---|
95* | 1X | 3:00 |
98 | 30X | 0:30 |
60 | 30X | 0:30 |
72 | 30X | 0:40 |
72 | 1X | 10:00 |
4* | 1X* | 0:00* |
*pause step
Extra PCR
Add 2.125 ul of the MM directly to the previous PCR
Reagent | ul/rxn | rxns | ul needed |
5x Iproof buffer | 0.425 | 272 | 185.3 |
10 mM dNTPs | 0.4 | 272 | 174.4 |
Primers | 1.33 | 272 | 580 |
Total | 2.155 | 272 | 939.7 |
Then continue with the last four unlisted steps for GBS1 from above:
Temp C | Cycles | Time |
---|---|---|
98 | 1X | 3:00 |
60 | 1X | 2:00 |
72 | 1X | 10:00 |
4 | 1X | 0:00 |
Pippin Prep Size Select:
Pool 2 ul from all wells of the 3 final plates into an 8 tube strip. Pool 30 ul from each of these into one tube after mixing via pipette.
Run Final Product on qPCR for check* (Do we still want to do this and the bioanalyzer?)
Result from qPCR check:
HPau_PP_Final:
The full result report can be viewed below:
Mail for sequencing:
Fill out the Shipping and Receiving domestic shipping form.
correct ORED shipping account number: 10-200-010002-70009-001-6023-0000-0
Sequencing Facility Address:
University of Colorado
Brian Woessner
Genomics Core, Bldg RC-2 Room 9400
12700 East 19th Ave.
Aurora, CO 80045
303-724-6050
email unsigned pdf to shipping.request@uwyo.edu by noon
Print, sign, and attach to package
Fill out Sequencing submission form
Insert inside package and email to brian.woessner@cuanschutz.edu
Make sure labeled tubes and ice packs are secure and bring down to Berry Center Office