The library pool was flagged by Psomagen as suspect. They could not get it to qPCR and the size was too small by approximately the extension accomplished via PCR2.
Enlarged Electropherogram from Psomagen
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1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | |
|---|---|---|---|---|---|---|---|---|---|---|---|---|
A | NTC | NTC | NTC | PE1 A1 Take 12 | PE1 A1 Take 21 | PE1 A1 Take 3 | PE1 A1 Take 4 | |||||
B | 0.0002 pM Std | 0.0002 pM Std | 0.0002 pM Std | PE1 B1 Take 12 | PE1 B1 Take 21 | PE1 B1 Take 3 | PE1 B1 Take 4 | |||||
C | 0.002 pM Std | 0.002 pM Std | 0.002 pM Std | PE1 C1 Take 12 | PE1 C1 Take 21 | PE1 C1 Take 3 | PE1 C1 Take 4 | |||||
D | 0.02 pM Std | 0.02 pM Std | 0.02 pM Std | PE1 D1 Take 12 | PE1 D1 Take 21 | PE1 D1 Take 3 | PE1 D1 Take 4 | |||||
E | 0.2 pM Std | 0.2 pM Std | 0.2 pM Std | PE1 E1 Take 12 | PE1 E1 Take 21 | PE1 E1 Take 3 | PE1 E1 Take 4 | |||||
F | 2 pM Std | 2 pM Std | 2 pM Std | PE1 F1 Take 12 | PE1 F1 Take 21 | PE1 F1 Take 3 | PE1 F1 Take 4 | |||||
G | 20 pM Std | 20 pM Std | 20 pM Std | PE1 G1 Take 12 | PE1 G1 Take 21 | PE1 G1 Take 3 | PE1 G1 Take 4 | |||||
H | PE1 H1 Take 12 | PE1 H1 Take 21 | PE1 H1 Take 3 | PE1 H1 Take 4 |
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Take 4: Fresh full prep using new dilutions of flow cell primers and original normalized template plate
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It looks like I did something wrong with my dilution of the flow cell primers. I should maybe do a test pcr before rolling with a new dilution. Both the completely redone pcrs and those redone from the original PCR1s gave good results within the expected range. Both, once adjusted for dilution were in the single to double digit nano molar range. I will proceed with redoing PCR2 from the saved PCR1s.