Fourth Week Blog

 Fourth Week Fun


    This week, we ran a second large gel holding a different set of samples from last week. We used the separate set hoping that it would show up on the gel, as the samples the previous week didn't show on the gel at all. After running the gel, not even primers showed up, which is why we ran the second set of samples, hoping that we would get results. We added 10.75 μl of mastermix, .5 μl FWD, .5 μl DNA, and 37.75 μl of PCR water. After loading the results on a gel, we let it run for about 2 hours. After running the gel again we got the same results as the previous week, however, it's very possible no primers we added to the samples. Another thing I did was learn more about enzyme restriction/ligation from Lizzy and Jonathon, and how they might change their protocol to help with the DNA's binding. 


New Protocol for Enzyme Restriction/Ligation

    1. Measure the DNA concentration of insert and vector plasmids using a spectrophotometer.

2. Add equal amounts of each insert and vector to the reaction mix. Use 20 fmol of each plasmid in a total reaction volume of 15 or 25 µl as follows:

For one to three fragments:

  • x1 µl vector DNA

  • x2 µl insert 1

  • x3 µl insert 2

  • x4 µl insert 3

  • 1.0 µl T4 DNA ligase

  • 0.5 µl type IIS enzyme

  • 1.5 µl 10× ligation buffer

  • H2O to 15 µl

3. Incubate the mix in a thermal cycler with the following conditions:

For one to three fragments:

  • 37°C for 2-4 hr

  • 50°C for 5 min

  • 80°C for 10 min

  • 16°C

Transform product into E. coli

4. Add the entire ligation mix (15 or 25 µl) to 50 µl competent E. coli cells and incubate on ice for 30 min.

5. Heat shock at 42°C for 90 s. Let cells recover on ice for 2 min.

6. Add 500 µl LB or SOC medium and shake cells for 45 min at 37°C in a ThermoMixer.

7. Plate on LB plates containing the appropriate antibiotic and X-gal. Incubate overnight at 37°C.

For cloning reactions with one to three inserts, plate 10-20 µl. Plate larger volumes for increasing numbers of inserts, because the number of colonies obtained will be lower. For example, plate 50 µl or more for constructs consisting of more than three fragments.


(Dragomir)


Theory

    So this week, considering we got no results after running two gels at first, we surmised it was most likely due to human error. After running left + tet in some PCR again, and ran it on a gel, we found the brightest bands at temps of 54 degrees Celsius and 34 degrees Celsius - though the band at 54 degrees was brighter than the one at 34. Lani and Ryan also diluted a bunch of samples of DNA to be sequenced later. With results at the end of the week, I'd say it was successful.


End of the week gel results at 54 and 34 degrees Celsius


Kieran 



Comments

Post a Comment

Popular posts from this blog

Third Week Blog

Image
Third Week Wonders     This week, I mostly worked with gels and PCR again. I worked with Lani's group on Tuesday to amplify left + tet. We wanted a 10.75 MM with 2 μl of DNA, 1  μl primer (x2 for both ends of the DNA), and filled the rest with PCR water to reach a total of 20 μl. While the PCR went on, I also helped with making some media for other bio classes. We started by adding a small glass tube into a second vial, then filling it about halfway with solution.  The first gel run on Tuesday. Gave exactly the readings we hoped for.  Plasmid Extraction     While I haven't done a plasmid extraction by myself yet, I can still go over the protocol and what it entails. The first step is to resuspend the pellet in 250 μl of resuspension solution. Mix it all together, then centrifuge it. Next, we add 250 μl of Lysis solution. You should mix well by inverting the tube 4-6 times. Next, you add 350 μl of the neutralization solution and mix immed...
Read more

First Week Blog

First Week Learning      Thi s week , I learned about PCR, gel electrolysis, overlap, and restriction enzymes. We did some amplified some left + tet, and tried to combine the left + tet with the right. Despite the group’s best efforts, we were unsuccessful with getting a good sample of left + tet + right. While the experiments were happening, however, I was busy learning how to do said experiments - so I’ll go over what I leaned.  Gel Electrolysis       I learned how to make a gel to read how many base pairs a sequence of DNA has. First, you figure out how dense you want the gel, based on what DNA you’re trying to read. Afterwards, you make the gel by combining agarose and TAE, boiling it for 5 minutes, then let it harden from a liquid to a  gel while you have a comb inserted in it to make wells. After the gel is hardened, you can load your sample(s) of DNA into the wells - loading the first or second well with your ladder. The ladder is used...
Read more