5/6-5/8 Gibson Assembly on the Full Plasmid

 To start the week we had put the 3-Way Insert Fragment and the PRAD 1 Vector into the Gibson master mix and let it incubate at 50C for 1 Hour. We then had to wait a couple days so that the confirmation primers were ready to use and once they were ready we put our Assembled Plasmid into two different PCR reactions. One reaction with the Chloramphenicol confirmation primer and the other with the Ampicillin conformation primer since the size difference between the primers meant that the extension time and annealing temperatures would be different. Once this PCR was done running, we had put this on a gel. With the 1st lane being the 1KB ladder, the 2nd lane was the Chloramphenicol confirmation primer (3503bp), and the 3rd lane was the Ampicillin confirmation primer (4681bp). The results initially were disappointing. We initially believed that the Gibson Plasmid did not work, however there could be a chance that in relation to all the side products the Gibson Plasmid has very low concentration. Also, the streaks that can be seen all throughout the lanes would suggest that there is some product there. So, when we are back in the lab we plan on using this sample in hopes that we can transform the DH5a E. Coli, as well as transform D. Rad with the 3-Way Insert Fragment that we also have. 

Ln 1: 1KB, Ln 2: Chlor Primer, Ln 3: Amp primer

 

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