Joe Yuan @ The Mason Lab: Post #3: Making a hybrid plasmid

It has been another fantastic two weeks of learning here at the Mason lab.

Over the past few weeks, I attempted to merge sections of two plasmids. And I shall attempt to illustrate this process with words:

So first, we know that we wanted a plasmid (end product) that will contain the DNA of the a pELxPS backbone, and the DNA of the canine CD19 we are trying to insert into canine K562 cells for the production of cells that produce cCD19. We know that the back bone and cCD19 sequences are at specific base-pair counts, and that they can be cut with a specific restriction enzyme. So we decided to employ a combination of restriction enzyme reactions and gel electrophoresis. By splitting segments of both pELxPs and cCD19, we were able to physically separate them by running gels and finding the bands at the correct base-pair count. 

Made the lab meeting worth it.

After isolating the 2 pieces of DNA we wanted, now we had to anneal them to each other, before proceeding to the amplification process commonly known as PCR. Luckily the backbone and functional region (cCD19) are naturally annealing, because our restriction enzymes are designed so that the end points of the two segments would align perfectly. Amazing! After the DNA has connected itself together, a PCR process was done to amplify the small yield from the previous experiment. After that, of course, we had to stick it into bacteria. A mini-prep was done to generate bacteria that had the DNA product we have generated. After plating it out on an LB agar plate with Ampicillin (AP Bio anyone?), it was left to grow before we chucked it back into high yield production.

Here's the part that I hate the most.

Maxi-preps.

This is a FIVE HOUR process, where spin cycles, filters, columns, and millions of 50ml conicals come together to create a "reasonable" amount of DNA, so that virus can be made with that DNA inserted. I had to do this 5 times already. It sounds as exciting as YouTube drama, and in reality it is actually less interesting than that, much less. So, lets fast forward.

Now we have spun 8 tubes of virus for 8 hours straight in a dying 20 year old centrifuge, and created lentivirus that will infect our cells, let me just dump that on 400,000 K562 cells. And what do you know. It worked. *sighs*

That was the past few weeks for you, and hopefully you are learning as much as I am.

"Gotta go get lucky".
~ Joe

Here's one for Sri & Meg.