Continue on overlap and restrictions of linear plasmid

 In this week's lab, we believe we had a mix-up on tubes we were working with when doing a heat gradient of left and tet we ran our overlap tubes that should not have primers and run on a gel if we see something then we know our tubes got mixed up. So we ran the tubes we thought got mixed up on a 1.2% gel but a bigger one that needs 120uL TAE buffer and 1.44g of agarose. After it was run on a gel we saw nothing at all so were not exactly sure what even happened so we decided to start all over again. We worked on a gel cleanup.


We used this kit for the gel cleanup. the instructions we followed were we cut out our DNA fragment from our agarose gel we made sure to trim the remaining excess gel. we then transferred that to a tube and weighed the gel slice. Then added 4 volumes of gel dissolving buffer to the gel slice. then incubate the sample between 37-55 C(typically 50C) inverting periodically until the gel slice is completely dissolved. Insert the column into the collection tube and load the sample onto the column. Spin for 1 minute, then discards the flow-through. Re-insert column into the collection tube. Add 200 μl DNA Wash Buffer and spin for 1 minute. Discarding flow-through is optional. We repeat the wash again Transfer the column to a clean 1.5 ml microfuge tube. Use care to ensure that the tip of the column has not come into contact with the flow-through. If in doubt, re-spin for 1 minute before placing into a clean microfuge tube. Add ≥ 6 μl DNA Elution Buffer to the center of the matrix. Wait for 1 minute, then spin for 1 minute to elute DNA.  The left mass of the gel is 192.9 buffer 771.6. Tet mass of gel is 248.3 buffer 993.2.

We then isolated left and tet to run on a gradient of 34-54 C. We also had a 9th tube called our hail mary left, tet, and right all in one tube at 44 C. After the gel cleans up we got 10.1 nanograms per ul for left and tet we got 21.6 nanograms per ul. we need to dilute left and tet but for left we did 15ul of sample from the 30ng/ul and 30 PCR dilution. Then tet 16 samples and 48 dilutions. in all our tubes we had 11ul master mix, 12ul DNA ( which is 5ul left and 7ul tet) 1 ul PCR h2o equals 24ul reaction and the hail mary mix is 11 master mix, 5 left, 5 right, and 7 tet. which we ran all 9 tubes on the 34c-54c gradient.

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. 


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