Continue on overlap and restrictions of linear plasmid and plasma extract

 This week for the lab we worked with our amplified sample of overlap. We made a 1.2% gel which is 3.6g of agarose and 30mL of TAE buffer. When we placed our product in the wells we used 10 ul of sample and 2ul of dye not using PCR water because we wanted a better view of what we had in our sample. 

In this photo, you'll see in the second row our 6ul ladder and our fourth row is the 12ul sample in the gel.

We ran that gel and it took the whole lab session so we worked on extracting plasma from Ecoli that was grown over the weekend. So we can have more tet since we already have a lot of left and right. We used the molecular biology Thermo scientific GeneJET Plasmid Miniprep kit. The website we used to follow the directions is thermofisher.com/studio first step was we resuspended the pelleted cells in 250ul of resuspension solution and pipetted up and down til we no longer had cell clumps. Then add 250ul of the lysis solution and 350ul of the neutralization solution, flipped upside down 4 times. Then we let it centrifuge for 5 min and got a white precipitate which we ran 3 times of 500ul solution. I didn't finish the rest but it was taken care of later by Leilani and then we got 303 anagrams which are concentrations of DNA and A260/A280 which is the ratio of DNA to proteins is 1.88. Then A260/ A230 is the ratio of salt to DNA is 2.33. Tuesday we isolated our Ana left and yet and right as well as Hilgarth left and tet and right. For Anna 10.75 ul master mix, 5ul forward primers, 5ul reverse primers, 5ul DNA, and 37.75ul PCR water which equals 50ul. For Hilgarth 1.5ul forward primer, 1.5ul reverse primer, 21.5ul Master mix, 23.5ul PCR water, and 2ul DNA equals 50 ul. 


In this picture we placed Ladder in well 1, space for well 2,  3 Anna Left, 4 Anna tet, 5 Anna right, 6 Hilgarth left, 7 Hilgarth tet, 8 Hilgarth right. We ran it 40 milliamps consent. 

We ran this gel before we wanted to do our great gel for a gel extract to see if we were going to see any bonds before we might possibly waste dye. luckily we did because we realized our Hilgarth was not good and we got ghost bands but our anna looked great. 

The last 3 wells are Hilgarth so you can see the ghost bands and Anna the 3 next to Hilgarth look clean and great. Since Anna is great we decided to focus on Anna and set Hilgarth aside since we wanted to do a gel excision we made a 1.2% gel with double wells so we were able to place more DNA so when we do the clean up we should have a lot left. so we did 45ul of Anna sample left, tet, and right and 9 ul of dye equals 54ul in our wells and 6ul of the ladder. 


in the image above the 1st well is our ladder, 3 wells are Anna's left, 4 Anna tet, and 5 are Anna's right.

Sources: 

1. Anna Behle 2019. Overlap extension PCR. protocols.io https://dx.doi.org/10.17504/protocols.io.psndnde

2. Hilgarth RS, Lanigan TM. Optimization of overlap extension PCR for efficient transgene construction. MethodsX. 2019 Dec 4;7:100759. doi: 10.1016/j.mex.2019.12.001. PMID: 32021819; PMCID: PMC6992990. Modified GCC Biotechnology 2022. 

3. Molecular biology Thermo scientific GeneJET Plasmid Miniprep kit


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