Continue on overlap and restrictions of linear plasmid

  This week for the lab we continued to work with 8 tubes that have been overlapped and amplified on a gel we used the raming left in our PCR tubes which were a 15ul sample. So we used the 15ul of sample and 3ul dye and ran it on a 1.2% gel. The recipe for a 1.2% gel is 120ml TAE buffer and 1.44g of agarose. 

In the image above you can see the ladder in our first well and then the third well is 1 which was running at 54 C and lastly, 8 ran at 34 C. from this image we were able to see 54 C which is the number run has a good pair of bands and the rest have little ghost bands. 

So another day we decided since we were getting a good result on the high gradient at 54 C we will run another heat gradient but the temperature will be raised higher at 52 C-70 C to make it overlap. When we were making our 8 tubes with 8ul of tet, 5ul of left, and 11ul of the master mix which equals 24ul in each tube we have 8 of. sadly when we were making our tubes we ran out of left so we were only able to disperse left in 1,2,3, and 4 but all 8 tubes got tet and master mix. So we did have to pause making our overlap tubes to make more tet and left to have enough. We also made 3 gels for the next day so when we're ready to run our overlap on a gel it would already be ready. we made 2 1.2% gel with double wells and one ain't gel.
Then the next day the team was able to put left in the 4 tubes that didn't get it and run them on a heat gradient of 54C-70C we need to amplify next. We did 10.75ul of master mix, 8ul of primer A, and 8ul primer D, and lastly 2.25*8 PCR water all in 1 tube. From that tube, we took 15ul and 5ul DNA from each heat gradient to make 8 more tubes that are amplified. after all 8 tubes were ready we ran them on a 62C gradient to amplify.  We then ran it on the big gel that we made the previous day.

The first well is our ladder, then we skip, and in the third well is 1 which was run at 70C through 8 which was run at 53C. each well had 5ul of the sample, 5ul PCR water, and 2ul dye. Once it was done running we checked to see if we were able to see bands and nothing surprisingly showed up were not 100% what went wrong but we will try something else once we get back from break.

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. 


Comments

Post a Comment

Popular posts from this blog

Continue on overlap and restrictions of linear plasmid and plasma extract

Image
 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 ups...
Read more

Continue on overlap and restrictions of linear plasmid

Image
 This week in lab we ran a heat gradient on right and tet. in the vary being we genuily believed we were not going to get an overlap at all and then we did which was so exciting for us until we concerned maybe some mix up happen. So on Monday our heat gradient was 35C-60C we've been having problems with our right so were hoping we can see anything with overlap. Our 8 tubes ran 1-8 listing the heat gradient below: 1- 60C, 2-57.9C, 3-54.6C, 4-50.1C, 5-44.5C, 6-39.6C, 7-36.7C, 8-35.0C In each tubes we had 11uL Master Mix, 7uL PCR water, 2uL right, and 4uL tet = 24uL in each tube(8). Also made a big gel 1.2% recipe is 120mL Tae buffer and 1.44 agarose. Once the heat gradient was done we ran it on a gel and these are the results: first well is ladder, space, and then 60C through 35C we realized we really like 8(35C), 7(36.7C), and 2(57.9C) so we ran the remaining which was 45uL and then added 9uL od dye and ran that on a gel so we could do a gel extraction. we also ran a PCR overlap 35C...
Read more