...
Size select for 300-366bp fragments size window via Pippin Prep
Sending Out for Sequencing
5% PhiX spike
Check In Samples Against List from Will Rosenthal
Load Submission Data into MISO
Quantify and normalize samples:
Normalize plate reader with TE for all plates
Quantify all plates
Normalize first 3 to 10 ng/ul and others to 30 ng/ul
Restriction Digestion
(Keep MM and reaction plates on ice)
Set Incubator to 37 C
Add 3 ul Digestion MM to 32 plates using the 8 channel
...
Reagent
...
ul/rxn
...
rxns
...
ul needed (1.5x)
...
10x T4 Buffer
...
1.15
...
600
...
690
...
5M NaCl
...
0.12
...
600
...
72
...
1 mg/ml BSA
...
0.6
...
600
...
360
...
H2O
...
0.73
...
600
...
438
...
MseI (enzyme)
...
0.12
...
600
...
72
...
EcoR1 (enzyme)
...
0.28
...
600
...
168
...
Total
...
3
...
600
...
1800
Add 6 ul template to each plate with Benchsmart. 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
...
600
...
600
...
H2O
...
0.112
...
600
...
67.2
...
10x T4 Buffer
...
0.1
...
600
...
60
...
5M NaCl
...
0.01
...
600
...
6
...
1 mg/ml BSA
...
0.05
...
600
...
30
...
T4 DNA ligase
...
0.1675
...
600
...
100.5
...
Total
...
1.4
...
600
...
840
Add 1 ul of EcoR1 Adaptors with 96-channel and Track which template plate gets which MID plate.
GTL Plate | EcoR1 MID plate | Pool Plate | Library | Norm to | Sequencer | |||
|---|---|---|---|---|---|---|---|---|
WCR49WCR57 | 1 | 1+2 | 1 | 10 | Nova | WCR50 | ||
2WCR58 | 1+2 | 110 | Nova | |||||
WCR51 | 3WCR59 | 3 | 1 | 10 | Nova | |||
WCR53 | 4 | 4+5 | 2 | 30 | Nova |
Label reaction plates with MID plate used.
...
Reagent | ul/rxn | rxns | ul needed (x1.4) | |
|---|---|---|---|---|
H2O | 9.52 | 350200 | 3094 | 25701904 |
5x iProof buffer | 4 | 350200 | 1300 | 1080800 |
10 mM dNTPs | 0.4 | 350200 | 13080 | 108 |
50 mM MgCl2 | 0.4 | 350200 | 130 | 10880 |
5 uM Illumina Primers | 1.33 | 350200 | 432266 | 360 |
iProof TAQ | 0.2 | 350200 | 6540 | 54 |
DMSO | 0.15 | 350200 | 4930 | 40.5 |
total | 16 | 350200 | 5200 | 43203200 |
Add 16 ul of PCR1 MM to each well of four new hard shell PCR plates with 8 channel
...
Reagent | ul/rxn | rxns | ul needed (x 1.6) | |
5x Iproof buffer | 0.425 | 360200 | 153 | 11585 |
10 mM dNTPs | 0.4 | 360200 | 14480 | 108 |
Primers | 1.33 | 360200 | 479 | 360266 |
Total | 2.155 | 360200 | 776 | 582431 |
Then continue with the last four unlisted steps for GBS1 from above:
...