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===Discussions=== #I terminated the PCR reactions at the end of 22th cycle when the amplification curves for the 7 reactions with gel came up whereas the NTC was still almost flat. This is a good indication that tagmentation worked to some extent. Based on the PAGE gel, there are faint smears in the 7 reactions. They are not strong enough, it's probably because the PCR reactions were terminated 1-2 cycles earlier. Overall this seems to be promising enough to proceed. #There are a few steps that could be improved in the future experiments. ##I wasn't very confident on the quality of cell lysis, because Alice got much better results with the other buffer using NP40. I did add fresh protease into this lysis buffer, and also used the buffer to lyse ~20,000 cells and checked with microscope. The cells seemed to be lysed, but I still think it worth trying the NP40-based buffer next time. ##On-gel bisulfite conversion is tricky, because it's hard to fill the FrameSeal chamber completely and prevent leaking during the high-temperature incubation. It is possible that the conversion rate is better in some area of the gel than the other. Covering the gel with coverslips and seal with mineral oil seems to be a better option. Definitely worth trying next time. ##I check the gel with microscrope at SYBG channel after MDA, but didn't see any signal except for some debris. I wasn't sure whether the MDA was working or not. However, if the amplification occurred evenly across the gel, I would not see anything either. Since I was able to get some amplicons after tagmentation/PCR, it's likely that MDA did work to some extent. ##There were a few bubble on the gel when I did in-gel tagmentation. Some small areas of the gel was damaged after the previous steps, such that the coverslip couldn't sit flat on the gel. One solution is to be more careful and try not to damage the gel. Another idea is to increase the volume of tagmentation reaction mix from 20ul to 25-30ul. ##Cutting gel into small piece and collecting the pieces individually is a little bit tricky. It's important not to do it when the gel is too dry. Transferring little gel pieces into PCR tubes is not easy either. I ended up pipetting 5ul of clean water to the scalpel tip that carries the gel piece, then transferring the water (with gel piece inside) to PCR tubes with the help of a P20 pipette.
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