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===Capture PCR=== * TBE Gel for capture at 56.4 (sample conditions), amplified 4 uL for 20 cycles. [[File:HOTSPOTS_captureA_E_newTemp.jpg | 300px]] * It is still unclear if the expected product was formed. There is a band slightly above 400 bp and another band slightly above 500bp. * B,E appears to perform the best (highest concentration of probes) * qPCR plot [[File:HOTSPOTS_capturePCR_A-E.bmp | 700px]] * Repeat PCR on A,C,D,and F for 21 cycles, and re-do gel with 8 ul loaded this time. I can now see the band at 471 (below 500bp and above 400bp). However, the capture is still inefficient. There are several ways to improve it, first, I can increase the amount of samples because here I've only used 150 ng while Noi usually use 200 ng. I can try with 300 ng. Also, F capture did not work because I either didn't amplify enough or need to use more probes. It appears that the quality of the individual subsets do vary substantially, but I'm not sure if this variation would be consistent across multiple captures. Also, even though annealing temperature 56.4 degC worked here, I wonder if there is a difference between 56, 56.4, and 56.8, so I might try to repeat using subset A at those temperatures. [[File:HOTSPOTS_capture56.4PCR_repeat | 300px]]
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