Continue on overlap and restrictions of linear plasmid

 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 and 63C we followed the same with  11uL Master Mix, 7uL PCR water, 2uL right, and 4uL tet = 24uL in 2 tubes that are sepretaly 1 running at 35C and 63C. 

After the gel was run and we were able to cut out the gel we needed we worked on the gel extraction. Following the Monarch gel extraction kit protocol card NEB# T1020 provided in the kit. i had to weight the tube with out the gel extraction and then with the gel and subtract those numbers to know what my gel weights. 

35C=  tube weighted 1356.6mg/ with the gel 1576.2mg

36.7C = tube weighted 1357.9mg/ with the gel 1632.9mg

57.9C= tube weighted 1371.9mg/ with the gel 1649.8mg

we then subtracted those numbers and found out exactly how much the gel weights

35C gel is = 219.6mg 

36.7C gel is 275mg

57.9C gel is 277.9mg 

then the next step stated is to add 4 volumes of gel dissolving buffer to the gel so we have to multiple the weight of the gel by 4 to know the exact amount each tempture needs in each tube. 

35C buffer is 878.4/ 36.7C buffer is 1,100/ 57.9C buffer is 1,111.6

then after the buffer was placed in the tube i need to incubate the sample at 55C til the gel is dissolved then i insert the liquid in another test tube and spin it for 1 min and waste any remaining liquid after repeating that step i move on to adding 9ul of DNA elution buffer. then we ran it on the nangram to see what our result are 

first result is 35C, then 36.7C, and lastly 57.9C

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