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.
Restriction Digestion
(Keep MM and reaction plates on ice)
Add 3 ul Digestion MM to 22 plates using the 8 channel
Reagent | ul/rxn | rxns | ul needed |
|---|---|---|---|
10x T4 Buffer | 1.15 | 450 | 517.5 |
5M NaCl | 0.12 | 450 | 54 |
1 mg/ml BSA | 0.6 | 450 | 270 |
H2O | 0.73 | 450 | 328.5 |
MseI (enzyme) | 0.12 | 450 | 54 |
EcoR1 (enzyme) | 0.28 | 450 | 126 |
Total | 3 | 450 | 1350 |
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.
Reagent | ul/rxn | rxns | ul needed |
|---|---|---|---|
MseI oligo | 1 | 440 | 440 |
H2O | 0.112 | 440 | 49.28 |
10x T4 Buffer | 0.1 | 440 | 44 |
5M NaCl | 0.01 | 440 | 4.4 |
1 mg/ml BSA | 0.05 | 440 | 22 |
T4 DNA ligase | 0.1675 | 440 | 73.7 |
Total | 1.4 | 440 | 633.38
|
Add 1 ul of EcoR1 Adaptors with 8-channel and Track which template plate gets which MID plate.
Template | EcoR1 MID plate |
|---|---|
2017_ALFALFA_PLATE10 | GBS Plate 5 |
2017_ALFALFA_PLATE11 | GBS 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 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 |
|---|---|---|---|---|
A | 2017_ALFALFA_PLATE10_Thermocycler | 2017_ALFALFA_PLATE11_Thermocycler |
|
|
B | 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 |
Add 16 ul of PCR1 MM to each well of two new hard shell PCR plates.
Add 4uL of template from pooled plates.
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 | 244 | 106.25 |
10 mM dNTPs | 0.4 | 244 | 100 |
Primers | 1.33 | 244 | 332.5 |
Total | 2.155 | 244 | 538.75 |
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 |
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 | 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 | 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 | 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 | 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 | 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 | 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 |
|
|
|
|
Combine 5uL from each well of GBS10-11_PCR_A into a pool. Make a 1:1000 dilution of this pool to run on qPCR.
Combine 5uL from each well of GBS10-11_PCR_B into a pool. Make a 1:1000 dilution of this pool to run on qPCR.
After 1:1000 pools have been made for each PCR plate, combine PCR_A and PCR_B undiluted pools together into one tube and label GBS10-11. Make a 1:1000 dilution of this pool to run on qPCR.
Add 16 ul of Illumina Library Quantification MasterMix to each well:
ul/rxn | Reagent | # of rxns | ul needed |
|---|---|---|---|
10 ul | KAPA SYBR FAST qPCR MM (2X) | 110 | 1100 |
2 ul | Primer Premix (10X) | 110 | 220 |
4 ul | Ultra Pure Water | 110 | 440 |
16 ul | Total Volume | 110 | 1760 |
Add 4 ul of template, pool, or standards to each well:
| 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 |
|
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Results:
Average quantities observed for all products:
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:
GBS10-11 PCR_B Pool:
GBS10-11 Pool:
GBS1-4 PCR_A Col_1:
GBS1-4 PCR_B Col_1:
Bioanalyzer
BioAnalyzer Chip Set up:
Well 1: GBS10-11 PCR_A Pool
Well 2: GBS10-11 PCR_B Pool
Well 3: GBS10-11 Pool
Well4: GBS10-11 PCR_A F3
Well 5: GBS10-11 PCR_A B5
Well 6: GBS10-11 PCR_A G7
Well 7: GBS10-11 PCR_A H12
Well 8: GBS10-11 PCR_B C1
Well 9: GBS10-11 PCR_B A4
Well 10: GBS10-11 PCR_B D6
Well 11: GBS10-11 PCR_B H8
Well 12: GBS10-11 PCR_B E11