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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We will repeat this one more time with the flow cell primers created anew from the 300 uM stock using one plate, PE1. We will also do a repeated PCR1 for this plate to include in the qPCR comparison.
1:1000 dilutions of final PCRs loaded.
1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | |
|---|---|---|---|---|---|---|---|---|---|---|---|---|
A | NTC | NTC | NTC | PE1 A1 Take 2 | PE1 A1 Take 1 | PE1 A1 Take 3 | PE1 A1 Take 4 | |||||
B | 0.0002 pM Std | 0.0002 pM Std | 0.0002 pM Std | PE1 B1 Take 2 | PE1 B1 Take 1 | PE1 B1 Take 3 | PE1 B1 Take 4 | |||||
C | 0.002 pM Std | 0.002 pM Std | 0.002 pM Std | PE1 C1 Take 2 | PE1 C1 Take 1 | PE1 C1 Take 3 | PE1 C1 Take 4 | |||||
D | 0.02 pM Std | 0.02 pM Std | 0.02 pM Std | PE1 D1 Take 2 | PE1 D1 Take 1 | PE1 D1 Take 3 | PE1 D1 Take 4 | |||||
E | 0.2 pM Std | 0.2 pM Std | 0.2 pM Std | PE1 E1 Take 2 | PE1 E1 Take 1 | PE1 E1 Take 3 | PE1 E1 Take 4 | |||||
F | 2 pM Std | 2 pM Std | 2 pM Std | PE1 F1 Take 2 | PE1 F1 Take 1 | PE1 F1 Take 3 | PE1 F1 Take 4 | |||||
G | 20 pM Std | 20 pM Std | 20 pM Std | PE1 G1 Take 2 | PE1 G1 Take 1 | PE1 G1 Take 3 | PE1 G1 Take 4 | |||||
H | PE1 H1 Take 2 | PE1 H1 Take 1 | PE1 H1 Take 3 | PE1 H1 Take 4 |
Take 1: Original Prep
Take2: PCR2 redone from original PCR1 with original flow cell primer dilutions
Take3: PCR2 redone from original PCR1 with new dilutions of flow cell primers from original 300 uM stock
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.