We are expecting little to no difference. So, we will proceed with actual template, but the smaller PCR pool. We will compare via qPCR and bioanalyzer.
Templates:
ALF_PLT10_2017_GBS | ALF_PLT11_2017_GBS |
Restriction Digestion
(Keep MM and reaction plates on ice)
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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 for 1 hour. Do 1 set on 2 thermocyclers and 1 set in incubator (Somebody will have to return in 8 hours).
Adaptor Ligation
Spin down plates and add 1.4 ul Ligation MM to each well with 8 channel.
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Template | EcoR1 MID plate |
|---|---|
2017_ALFALFA_PLATE10 | GBS EcoR1 Plate 5 |
2017_ALFALFA_PLATE11 | GBS EcoR1 Plate 6 |
Label reaction plates with MID plate used.
Cover, vortex, and spin plates. Incubate thermocycler plate at 16C for 6 hours. Incubate incubator plate at RT for 2 hours.
Add 189 120 ul of low EDTA TE store at 4C for a month or -20C for longer.
PCR Amplification
Create the two pools below using 20uL from ligated plates matching well to well.
Pool | Plate1 | Plate2 | Plate3 | Plate4 |
|---|---|---|---|---|
A2A | 2017_ALFALFA_PLATE10_Thermocycler | 2017_ALFALFA_PLATE11_Thermocycler |
|
|
B2B | 2017_ALFALFA_PLATE10_Incubator | 2017_ALFALFA_PLATE11_Incubator |
PCR1:
Reagent | ul/rxn | rxns | ul needed |
|---|---|---|---|
H2O | 9.52 | 220 | 2094.4 |
5x iProof buffer | 4 | 220 | 880 |
10 mM dNTPs | 0.4 | 220 | 88 |
50 mM MgCl2 | 0.4 | 220 | 88 |
5 uM Illumina Primers | 1.33 | 220 | 292.6 |
iProof TAQ | 0.2 | 220 | 44 |
DMSO | 0.15 | 220 | 33 |
total | 16 | 220 | 3520 |
...
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
...
Temp C | Cycles | Time |
|---|---|---|
98 | 1X | 3:00 |
60 | 1X | 2:00 |
72 | 1X | 10:00 |
4 | 1X | 0:00 |
Comparisons
qPCR
Make 1:1000 dilutions of column 3 from the PCR plates by adding 1 ul to 999 ul TE in a deep well plate:
...
| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 |
|---|---|---|---|---|---|---|---|---|---|---|---|---|
A | GBS10-11 PCR_A Col_1 | GBS10-11 PCR_A Col_6 | GBS10-11 PCR_A Col_12 | GBS10-11 PCR_B Col_1 | GBS10-11 PCR_B Col_6 | GBS10-11 PCR_B Col_12 | GBS10-11 PCR_A Pool | GBS1-4 PCR_A Col_1 | GBS1-4 PCR_B Col_1 | NTC | NTC | NTC |
B | GBS10-11 PCR_A Col_1 | GBS10-11 PCR_A Col_6 | GBS10-11 PCR_A Col_12 | GBS10-11 PCR_B Col_1 | GBS10-11 PCR_B Col_6 | GBS10-11 PCR_B Col_12 | GBS10-11 PCR_A Pool | GBS1-4 PCR_A Col_1 | GBS1-4 PCR_B Col_1 | 0.0002 pM Std | 0.0002 pM Std | 0.0002 pM Std |
C | GBS10-11 PCR_A Col_1 | GBS10-11 PCR_A Col_6 | GBS10-11 PCR_A Col_12 | GBS10-11 PCR_B Col_1 | GBS10-11 PCR_B Col_6 | GBS10-11 PCR_B Col_12 | GBS10-11 PCR_B Pool | GBS1-4 PCR_A Col_1 | GBS1-4 PCR_B Col_1 | 0.002 pM Std | 0.002 pM Std | 0.002 pM Std |
D | GBS10-11 PCR_A Col_1 | GBS10-11 PCR_A Col_6 | GBS10-11 PCR_A Col_12 | GBS10-11 PCR_B Col_1 | GBS10-11 PCR_B Col_6 | GBS10-11 PCR_B Col_12 | GBS10-11 PCR_B Pool | GBS1-4 PCR_A Col_1 | GBS1-4 PCR_B Col_1 | 0.02 pM Std | 0.02 pM Std | 0.02 pM Std |
E | GBS10-11 PCR_A Col_1 | GBS10-11 PCR_A Col_6 | GBS10-11 PCR_A Col_12 | GBS10-11 PCR_B Col_1 | GBS10-11 PCR_B Col_6 | GBS10-11 PCR_B Col_12 | GBS10-11 Pool | GBS1-4 PCR_A Col_1 | GBS1-4 PCR_B Col_1 | 0.2 pM Std | 0.2 pM Std | 0.2 pM Std |
F | GBS10-11 PCR_A Col_1 | GBS10-11 PCR_A Col_6 | GBS10-11 PCR_A Col_12 | GBS10-11 PCR_B Col_1 | GBS10-11 PCR_B Col_6 | GBS10-11 PCR_B Col_12 | GBS10-11 Pool | GBS1-4 PCR_A Col_1 | GBS1-4 PCR_B Col_1 | 2 pM Std | 2 pM Std | 2 pM Std |
G | GBS10-11 PCR_A Col_1 | GBS10-11 PCR_A Col_6 | GBS10-11 PCR_A Col_12 | GBS10-11 PCR_B Col_1 | GBS10-11 PCR_B Col_6 | GBS10-11 PCR_B Col_12 |
| GBS1-4 PCR_A Col_1 | GBS1-4 PCR_B Col_1 | 20 pM Std | 20 pM Std | 20 pM Std |
H | GBS10-11 PCR_A Col_1 | GBS10-11 PCR_A Col_6 | GBS10-11 PCR_A Col_12 | GBS10-11 PCR_B Col_1 | GBS10-11 PCR_B Col_6 | GBS10-11 PCR_B Col_12 |
| GBS1-4 PCR_A Col_1 | GBS1-4 PCR_B Col_1 |
|
|
|
Results:
Average quantities observed for all products:
GBS10-11 PCR_A Col_1: 78.13 nanomoles (thermocycler)
GBS10-11 PCR_A Col_6: 85.33 nanomoles (thermocycler)
GBS10-11 PCR_A Col_12: 75.57 nanomoles (thermocycler)
GBS10-11 PCR_B Col_1: 96.37 nanomoles (incubator)
GBS10-11 PCR_B Col_6: 78.72 nanomoles (incubator)
GBS10-11 PCR_B Col_12: 80.08 nanomoles (incubator)
GBS10-11 PCR_A Pool:Pool: 95.64 nanomoles (thermocycler)
GBS10-11 PCR_B Pool: 110.7 nanomoles (incubator)
GBS10-11 Pool: 95.91 nanomoles
GBS1-4 PCR_A Col_1: 87.22 nanomoles (repeated from previous qPCR)
GBS1-4 PCR_A Col_1:GBS1-4 PCR_B Col_1:B Col_1: 81.87 nanomoles (repeated from previous qPCR)
The full result report can be viewed below:
| View file | ||
|---|---|---|
|
Bioanalyzer
BioAnalyzer Chip Set up:
Well 1: GBS10-11 PCR_A Pool
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Well 12: GBS10-11 PCR_B E11
Gel:
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Electropherogram:
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EPG Pools:
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EPG Wells 4-8:
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EPG Wells 9-12:
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