Week 4 2/12-2/16: PCR Cleanup and Success with the pRHAM Fragment

 So I feel that for the Week 4 the best way to summarize this week (as well as week 5) as not too much had occurred during both weeks. But week 4 did yield great results as we were finally able to obtain a clean and viable pRHAM fragment for Gibson Assembly. So the week started out by doing two simultaneous yet separate gel electrophoresis’ for D. Rad PCR product and the pRHAM plasmid product which took a majority of the day and didn’t allow for much else to be done with for that day. In the next day, we ran PCR on our pRHAM plasmid as both gels had yielded the desired results and had both shown up on the gel as expected. So, while we ran PCR on the pRHAM plasmid we tried doing the PCR Cleanup protocol on the D. Rad pcr poducts while trying to figure out what went wrong with our prior D. Rad left fragments and how we can avoid making the same mistakes. So, after discussing the issue, we decided to carry out the protocol as normal, except we would isolate each waste into their own tube, as well as trying to increase our elution buffer to see how more elution buffer would effect our yield, and hopefully increase our 280/230 (impurities). So our results ended with:



MC represents our sample that we used, then our nanodrop readings. What we are specifically looking for is a 260/280 of 1.80-2.00 and a 260/230 of 1.8-2.00. If the values are around here then it means that our samples have little impurity and has a lot of DNA information that we need. As you can see, our sample practically fell apart after we had a total of 18 uL of elution buffer. We start with 6 uL of elution buffer since that is the minimal value that the protocol calls for, then we added an addition 6 uL to see the impact of adding more elution buffer. After adding more elution buffer (marked by the 12 uL) our sample yield had decreased significantly, our 260/280 decreased, but our 260/230 increased closer to the desired value. However, once was added another 6 uL (18 uL total) all of our numbers were completely throw off because there was such little sample left. So this told us that adding more elution buffer does increase our desired results, however if we add too much then our sample is at risk of being washed out. 

Next we tried the PCR Cleanup on our PCR product for the pRHAM once it was complete. Our results yielded: 


So, after our first cleanup we had actually received the results that we were looking for. We are unsure if this was because after we ran this protocol the first time that we had made some error, but we were able to yield a desired result from our first PCR cleanup protocol on the pRHAM PCR product. 


Comments

Post a Comment

Popular posts from this blog

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...
Read more

Week Three: 2/5 - 2/9 Setbacks and Pushing Forward

 Monday Where we left off the week prior we had done PCR cleanup procedure on our 4 PCR products, those being: D. Rad left flank, D. Rad right frank, pRAD 1 Vector, and pRHAM kanamycin resistant plasmid. The pRHAM PCR product and the D. Rad left flank PCR product did not yield good results after the PCR cleanup protocol, so on Monday we thought that we would retry the PCR cleanup kit on just those two PCR products to hopefully get better results. Unfortunately after doing the PCR cleanup protocol the results were the same as the week before with the products having low absorbance and low yield. With these results we had to redo a few steps in order to hopefully get better results. Throughout the week we would have to redo the plasmid extraction of the pRHAM plasmid, as well as regrow D. Rad, do another genomic extraction, and do PCR on the extraction genomic product. So, on Monday after we planned our next steps, we prepped 5 flasks of 25 mL of LB Broth which we could use to contin...
Read more

Week Two: 1/29 - 2/2 Gel Electrophoresis and PCR

Image
 Monday To start out the week we did Gel Electrophoresis on the plasmid product from the plasmid extraction that we did the previous Friday, 1/26. We did electrophoresis on the pRHAM plasmid product. Since electrophoresis is useful in identifying compounds based on their size in base pairs. Since DNA is negatively charged electrophoresis attracts the DNA from the negative side to the positive side of the gel. Compounds that have more base pairs move slower than compounds with less base pairs, so it useful to use a ladder as a reference point to how big the band should be. Our compound was around 2,300 base pairs so we used a 1 KB ladder that would separate the bands by around a thousand. Once the bands had ran far enough that we could get a good range of their size we turned off the machine and put the gel under UV light to see the bands more clearly. The band ended up being in the area between 2,000-3,000 base pairs which is what we were looking for, meaning that our extracted pro...
Read more