Test Plates
BS_PLT1 _Norm (4 samples)BS_PLT2_Norm (4 samples)Well C1 Normalized to 10ng/ul
PCR MasterMix
ul/rxn | Reagent | # of rxns | ul needed |
|---|---|---|---|
3 | 5X Kapa HiFi Buffer | 80 | 240 |
0.45 | 10M dNTPs | 80 | 36 |
0.3 | Kapa HiFi HotStart DNA Pol | 80 | 24 |
7.25 | HPLC H2O | 80 | 580 |
11 | Total Volume | 80 | 880 |
Add 11 ul to each well of a hard shell, full skirt plate. Add 2 uL of primers and 2uL of template to each well.
Primers: _______________ (fill in when primers arrive).AMF06
Template Format:
| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 |
|---|---|---|---|---|---|---|---|---|---|---|---|---|
A |
BSP1 C1 |
BSP1 C1 |
BSP1 C1 |
BSP1 C1 |
BSP1 C1 |
BSP1 C1 |
BSP1 C1 |
BSP1 C1 |
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B |
BSP1 C1 |
BSP1 C1 |
BSP1 C1 |
BSP1 C1 |
BSP1 C1 |
BSP1 C1 |
BSP1 C1 |
BSP1 C1 |
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C |
BSP1 C1 |
BSP1 C1 |
BSP1 C1 |
BSP1 C1 |
BSP1 C1 |
BSP1 C1 |
BSP1 C1 |
BSP1 C1 |
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D |
BSP1 C1 |
BSP1 C1 |
BSP1 C1 |
BSP1 C1 |
BSP1 C1 |
BSP1 C1 |
BSP1 C1 |
BSP1 C1 |
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|
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E |
BSP1 C1 |
BSP1 C1 |
BSP1 C1 |
BSP1 C1 |
BSP1 C1 |
BSP1 C1 |
BSP1 C1 |
BSP1 C1 |
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F |
BSP1 C1 |
BSP1 C1 |
BSP1 C1 |
BSP1 C1 |
BSP1 C1 |
BSP1 C1 |
BSP1 C1 |
BSP1 C1 |
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G |
BSP1 C1 |
BSP1 C1 |
BSP1 C1 |
BSP1 C1 |
BSP1 C1 |
BSP1 C1 |
BSP1 C1 |
BSP1 C1 |
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H |
BSP1 C1 |
BSP1 C1 |
BSP1 C1 |
BSP1 C1 |
BSP1 C1 |
BSP1 C1 |
BSP1 C1 |
BSP1 C1 |
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Run on thermocycler program BSP*:
Step | Temp C | Cycles | Time |
|---|---|---|---|
Denature | 95 | 1X | 10:00 |
Annealing | 94 | 35X | 0:30 |
Annealing** (Row E) | 55 | 35X | 0:30 |
Extension/Elongation | 72 | 35X | 1:00 |
Extension/Elongation | 74 | 1X | 9:00 |
Hold | 4 | 1X | 0:00 |
*BSP program will test the efficiency of the PCR/annealing temperature by running it on a temperature gradient for each row. The table below shows the annealing temperature for each row.
**
Row | Annealing Temperature |
|---|---|
A | 63 |
B | 61 |
C | 59 |
D | 57 |
E | 55 |
F | 53 |
G | 50 |
H | 48 |
MagBead Cleanup:
Some plates were cleaned up using the Nimbus platform protocol “AxyPrep MagBead PCR1 No MM”
...
Make 1:1000 dilutions of column 1,2,3 and 6,7,8 from the PCR plates by adding 1 ul to 999 ul TE in a deep well plate: (Will fill in with proper sample names.)
| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 |
|---|---|---|---|---|---|---|---|---|---|---|---|---|
A | S1_A | S2_A | S3_A | S4_A | S5_A | S6_A | S7_A | S8_A |
| NTC | NTC | NTC |
B |
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| 0.0002 pM Std | 0.0002 pM Std | 0.0002 pM Std |
C |
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| 0.002 pM Std | 0.002 pM Std | 0.002 pM Std |
D |
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| 0.02 pM Std | 0.02 pM Std | 0.02 pM Std |
E |
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| 0.2 pM Std | 0.2 pM Std | 0.2 pM Std |
F |
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| 2 pM Std | 2 pM Std | 2 pM Std |
G |
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| 20 pM Std | 20 pM Std | 20 pM Std |
H |
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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 | S1_A | S2_A | S3_A | S4_A | S5_A | S6_A | S7_A | S8_A |
| NTC | NTC | NTC |
B |
|
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|
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| 0.0002 pM Std | 0.0002 pM Std | 0.0002 pM Std |
C |
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| 0.002 pM Std | 0.002 pM Std | 0.002 pM Std |
D |
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| 0.02 pM Std | 0.02 pM Std | 0.02 pM Std |
E |
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| 0.2 pM Std | 0.2 pM Std | 0.2 pM Std |
F |
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| 2 pM Std | 2 pM Std | 2 pM Std |
G |
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| 20 pM Std | 20 pM Std | 20 pM Std |
H |
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Results:
Average results:
Temp | Avg uM |
|---|---|
63 | 6.95 |
61 | 9.64 |
59 | 10.36 |
57 | 10.9 |
55 | 10.29 |
53 | 10.46 |
50 | 10.22 |
48 | 8.99 |