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 continue growing the E.Coli that had the pRHAM plasmid. We also prepped 250 mL of TGY so that we could start regrowing D.Rad for the genomic extraction later in the week. Then we poured 4 plates of LB agar and added ampicillin to it so that we had plates ready to for for when we wanted to grow the pRAD1 plasmid E.Coli, since the pRAD1 E.Coli is ampicillin resistant. Finally, we streaked a TGY plate with D.Rad that we had from freezebac in the hood, following proper procedure to make sure everything was sterile. 

Tuesday

On Tuesday I was unable to stay long in lab, but there was not too much that we could do since many of our stuff was still growing. However, we did prep 4 flasks with 25 mL of the TGY broth that we had prepped the day before. But since there was not much that we could do, we had to wait for the next day to continue our plans. 

Wednesday

We started off Wednesday by making three freezebacs of the pRHAM plasmid E.Coli incase we needed this E.Coli later on and didn’t have any broths or plates of it. Once we had made these freezebacs we had them put in the -80 C freezer to stay until we needed them. During this time we wanted to do a plasmid extraction with the pRHAM plasmid, however there was an issue with the Neutralizing Buffer so while this was getting sorted out I made a 1% agarose gel that we can use to run the plasmids on once we had done the extraction. However, by the time a suitable Neutralizing Buffer was found it was too late and we could not do the plasmid extraction, so I wrapped the gel in cling wrap and put it in the fridge to be used the next day. We had also found the products of the genomic extraction that we had done a couple months prior in the -20 C freezer, so instead of having to redo genomic extraction, we could use these and just run PCR on them again. 

Thursday

While the primers were being hydrated for us to do PCR on the genomic extraction products, we decided to do plasmid extractions on the pRHAM plasmid. We decided to do 4 extractions of the pRHAM plasmid to hopefully get at least one good plasmid extraction. After we had done the extraction, the primers were ready and we started PCR on the genomic extraction, mixing the extracted genomic material with the primers that specifically bind to the left flank that we want. This would give us 4 left fragments after the PCR was complete. During PCR we wanted to run the plasmid product on the gel we had prepped the day before, however there wasn’t enough time and eppendorf tubes for us to mix the dye and plasmid with. So, we had run the plasmid through the nanodrop and on the next Monday we would run the plasmids and the PCR products on gels. While the nanodrop readings were not the best, we were told to still run them on a gel in hopes of having a a band show up around the expected range of 947 base pairs. We were told that there could have been low yield since the broth that we used to extract the plasmid was a little old by then and the E.Coli could have broken down the kanamycin resistance and had expelled the plasmid. 


Overall this week was definitely a setback from what we were expecting to do, and while it was unexpected we were able to push through it to make sure that we have the best pcr products that we can in order for our Gibson Assembly to hopefully work in the coming week or so. 

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