final overlap and restrictions of linear plasmid

 This week for lab I first worked on a plasmid extraction 1. At least 3 mL culture cells isolated.

  1. Do 1 mL of culture at a time.
  2. Spin down for a minute and pack pellet each time.

2. Wash w/ 500 uL multibuffers (×2).

  1. Mix by pipetting up and down.
  2. Let sit for 5 minutes.
  3. Spin down for a minute.

3. 600 L of 1mg/UL of lysozyme @ 37 °C for 15 minutes.

  1. Also pipette up and down to mix, no vortexing.
  2. DO NOT spin.

4. Then, add 100 L of 7X Lysis Buffer (Blue) and mix by inverting the tube 4-6 times. Proceed to the next step within 2 minutes.

a. DO NOT spin.

5. Add 350 ML of cold Neutralization Buffer (Yellow and mix thoroughly. The sample should turn yellow when neutralization is complete. A precipitate will form.

a. Invert an additional 2-3 times.

  1. Centrifuge for 2-4 minutes.
  2. Transfer supernatant (800 ML at a time) in spin column. Spin down for 1 minute and empty flow-through.

a. May need to do multiple times.

Add 200 ML of Endo-Wash Buffer to column. Centrifuge for 30 seconds.

a. No need to empty collection tube.

9. Add 400 ML of Zyppy Wash Buffer to the column. Centrifuge for 1 minute

10. Transfer the column unto a clean 1.5 mL centrifuge tube and then add 30 ML of Zypy Elution Buffer and let stand for 1 minute at room temperature.

11.Centrifuge for 30 seconds.

12. Nanodrop/Fluorometer for results

We got good result and ended with picture below shows results 





Then we ran a heat gradient of overlap running st 39C til 64C we worked on amplify the heat gradient to see if were able to get a full 3 way assembly the heat gradient was 
1-64C
2-61.9C
3-58.7C
4-54.1C
5-48.4C
6-43.7C
7-39C


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 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...
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