Taylor Brown Blog 4

Hello Everyone!

Sorry I’m late on my update haha; my labwork picks up and slows down, and now it’s slowed down, so I can write! Anyway, we got the sequencing results back! And I realized you may not know what colony PCR or a “miniprep” is…colony PCR is basically picking bacterial colonies and putting them, along with other reagents, into a machine that amplifies your DNA construct so that you can make sure it’s present by “running it on a gel” (gel electrophoresis). If you see bright bands around the size that your DNA should be, your DNA is there! A miniprep is basically a procedure you do after you have confirmed your DNA construct is present; you basically isolate the bacterial DNA from your colonies and purify it so that it can be sequenced. “Sequencing” is when you send your DNA off to a company so that they can provide you with the sequence of nucleotides (a long list of A,T,C, and G’s that code for your gene), and you compare your DNA results to the correct sequence that you were aiming for. Basically, the comparison looks like this:

 

except it’s much longer. The “subject” lines are part of the sequence for the gene that I want to study. The “query” lines are the sequencing results. You want a high Identity percentage, a low gaps percentage, and when you look at the long sequence you want to see lots of the vertical lines matching the letters in the subject and query lines (and no blanks where the vertical lines should be). So, my miniprep sequencing went well, and then it was on to a maxi prep! A “maxi prep” is basically like a miniprep, but growing up a LOT more bacteria. You start by taking a little bit of the bacteria that you used to do the colony PCR and the miniprep sequencing, placing it into LB broth (basically a liquid form of all of the nutrients the bacteria need to grow that looks like this:

(Photo credit to gbiosciences.com) and growing up a LOT of bacteria so that you can get a lot of DNA to work with (because as you know, bacteria replicate quickly). You know when you have enough a lot of bacteria because the flask gets really cloudy and smeeellllyyyyy! Haha I can’t describe the smell to you, really, but maxipreps aren’t my favorite thing to do; but they must be done! Anyway, then I purify the DNA again (with a procedure that I won’t go into detail about) and send it to sequencing one more time to confirm that I have the right DNA, and then comes the transfection! “Transfection” is when you take your DNA and viral components (the latter found in a liquid form in small tubes) and pipet them onto packaging cells in a dish. These cells then make the viruses for you with your DNA of interest, and then you put the virus onto the cells you really wish to study (called viral transduction), so that the virus can insert your DNA into your target cells. (My target cells are human breast epithelial cells called MCF 10As). But the whole process doesn’t just happen in a day, and it fails sometimes, so that’s what I’m working on now.

When you do virus work, you have to put on a space suit! Just kidding…but you have to put on a bunch of extra PPE (personal protective equipment) that includes: two pairs of gloves, a plastic gown with holes for your thumbs, and a face mask in addition to your labcoat (and long pants, socks, and close toed shoes). I modeled the process a bit for you below :) haha



And here's a picture of me in action :)



But anyway, looks like my cloning was a success guys! My mentor has another cloning project for me so that I can practice it some more, but looks like my first round of cloning went well! And I’m hoping my viral transfection and transduction worked so I can move on to my experiments! Thanks for reading; more info to come later!

Tay<3 br="" style="mso-special-character: line-break;"> p.s. …it’s my old man’s birthday today. Happy Birthday Papa! I love you(: