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

 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 product was good.

Plasmid Electrophoresis 

1KB Ladder Reference. Link to Page

Tuesday

On Tuesday we did PCR on the plasmid product since now we knew that our plasmid product from the previous week was good. For PCR we only had to do short amp since the fragment size that we wanted was 947 base pairs rather than being several thousand base pairs long. So, we started the PCR machine and during the 1.5 hours that the PCR machine was going we made another 1% agarose gel to run electrophoresis on the PCR product once it was out. This time we were looking for a fragment at around 947 base pairs so we used a 100 base pair ladder to get more precision on fragment sizes less than 1,000 base pairs long. Once the PCR finished we started up the electrophoresis soon after. The gel didn’t take too long to run and once it was done we went and put it under UV light once again. A fragment was seen at in between the 900 base pair and 1,000 base pair band which is what we wanted to see. 

100 Base Pair Ladder. Link to Page

PCR Product Electrophoresis

Wednesday

Wednesday was a less eventful day, we thought that we were going to start Gibson Assembly but there were specific calculations that we had to do before we could start, calculations that I did not know how to even start. So, I had to find the nano drop readings of all of our PCR products so far, including the pRHAM one that we had just done, the pRAD1 plasmid that we had done in November, and the D.Rad once that we had done in early December. Once I found the readings and the size of each fragment I was somewhat able to start doing the calculation but one last thing popped up before we could truly start the calculation, PCR cleanup. In PCR there are side reactions that can occur and we do not want any of those side products possibly messing with our Gibson Assembly protocol once we start it, so we had to do a PCR cleanup protocol for our PCR products to isolate the PCR product since that is the only thing that we care about. Since we had ran out of time on Wednesday we decided to do the PCR cleanup kit protocol the next day. 

Thursday

On Thursday we started the PCR Cleanup kit protocol on our PCR products. Since we had at least two products of every reaction we did the protocol on one of every product just incase something went wrong during the protocol. It took us a little time to figure out how much of certain solutions to add since the protocol requires 20-100 uL of the PCR product but we had at most 5 uL of some products and 2 uL of other products, so clearly we didn’t have enough of the PCR product required, But, the protocol says to add TE Buffer to get to the desired volume to start, but the kit doesn’t come with TE Buffer so we had to ask to get some TE Buffer. Once we had all of that figured out we were finally able to start the cleanup kit protocol. Once we were done, we ran the final products on the nano drop to see our results. Two of the cleanup products had good results and the other two products didn’t have the desired results. So, on Monday we are going to do the cleanup kit protocol on the two PCR products that didn’t have good results to hopefully get better results. 


NEB PCR Cleanup Kit Protocol: Biolabs, N. E. (n.d.). Protocol for DNA cleanup and concentration using the Monarch® PCR & DNA cleanup kit (5 μg) (NEB #T1030). NEB. https://www.neb.com/en-us/protocols/2015/11/23/monarch-pcr-and-dna-cleanup-kit-protocol  

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