Week Five: 2/19 - 2/23: Alcohol Precipitation Preparation, and D. RAD PCR

 So, due to the consistent undesired products from trying the PCR cleanup protocol on the Left Fragment of the D. Rad, we decided that it was best to try a different procedure that would similarly clean and isolate the desired product from our PCR product. The other protocol that we could try was Alcohol Precipitation utilizing ethanol rather than isopropanol since isopropanol specialized in bigger sample sizes which is something we did not have. Alcohol precipitation is very good at isolation and cleaning the fragment. The trade off for this procedure is that while it offers a better quality product, our sample size decreases significantly. This, however, isn’t that bad for us because in Gibson Assembly we are working with sample sizes that are smaller than microliters, so all we needed was a quality product over a large sample size. Our current issue then became the fact that we did not have much sample left from our D. RAD PCR. Our PCR leaves us with 20 uL max of sample, then we use 2 uL to nanodrop and make sure that our sample came out the way that we wanted it, and finally we would run a gel which we would use 10 uL of sample for it. This would leave us 12 uL down and at 8uL of sample left, at best. For future use, we were advised to only use at most 5 uL of our sample or 25% of the sample that we have, rather than the 50% that we were doing before, which would give us more product to work with. So, we ran a 50 uL PCR on our D. RAD left fragment, which allowed for us to nanodrop and have left over sample for the alcohol precipitation, the only issue would be that we have only one chance of doing this since it uses a lot of Master Mix (templates/primers) to perform PCR at such a high volume. So, next week (2/26 - 3/1) we will be attempting alcohol precipitation on our new PCR D.RAD left fragment samples. 

Sadly, I wasn’t able to spend as much time in lab this week as I would have liked but I will make up for it next week and hopefully we get good results and can do Gibson Assembly next week. 

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