I'm pushing myself to defend before July 30th, because that's graduation. I took a little time off of lab because I had a lot of personal things going on that I needed to sort out first, and now I'm back and determined to do what I can to blow through this thesis since the bulk of the work is already there.
In a nutshell, my project is about Prostate Cancer. Castration-Resistant Prostate Cancer (CRPC) is a phenomenon, so any clues I might be able to provide about CRPC cells could provide another piece of the puzzle in one big mystery.
One of the things giving me hope about making it is my planning skills.
I set up sooooooo many backups in case things went wrong for the project. I can't really control how WBs or PCRs go, but I can make sure the cells I collect to analyze were meticulously cared for so that the data I get is the data I'm confident it should be. Plan for the unplanable, they say. I can't tell you how many times I've saved my project because I had safeties in place (actually, I probably can--I kept track of EVERYTHING in my lab notebook). The hardest thing about what I'm doing is that it's all in vitro and the whole project is time sensitive and takes about a month each time, so I can't rush it. I knew ahead of time that it might be down to the wire like this, so the best I can do is account for growth times before I can begin the experiments, and then set up safety/contingency plans (like preparing a "back up" charcoal-treated FBS conical in case I'd need it many months later, pictured below).
|Prepared this for myself *just in case* back in October--yup! It's June & I needed it the other day to make new media|
Today is Day 0 (control) of a mini-repeat of the larger thing I did a while back.
I like planning ahead to figure out how much inventory I'd need to get through whatever I need to get through because budget, time, resources, and sharing the lab with others need to be accounted for.
Yesterday, I made myself 2 new bottles of media: 1 of "regular" FBS media (RPMI 1640 with L-glutamine and phenol red), and 1 "Charcoal-treated" media (RPMI 1640 with L-glutamine
and without phenol red). You need to let the media set for a day or two before you can use it because it'll get super bubbly. I already had back up bottles from last time, so now I don't have to stress about running out of media for my cell culturing. It's one less thing to worry about.
I decided a nice way of organizing what I'm doing (besides the lab notebook) and being able to do a little blogging is to keep track of what I'm doing each day in lab until I finish. It's pretty much a Lab Diary from this point on.
If you would like to follow my progress, please subscribe to my blog and join me!
12 Plates total
1 plate from each will serve as D0 (control plates/cells)
[x] Took Cell Photos @ 4x, 10x, 20x, 40x (in black & white)
[x] Trypsinized (2mL Trypsin, 8mL FBS. Transfer to 15mL conical. Spin @ 3k for 5 mins)
[x] Resuspend (Aspirate out old media, save pellet at bottom of conical, resuspend in 3mL FBS)
[x] Cell Count (Mix & take 20 uL for 1:1 ratio with Trypan blue to stain slide & count via Countess)
[x] Collect Step 1 (Spin again @ 3k for 5 mins. Aspirate out all but 1mL, careful not to touch pellet. Transfer 500uL to eppendorf tube for Western Blots & 500uL to eppendorf tube for RT-PCR)
[x] Collect Step 2 (Spin in 4 degree fridge @2500k for 5 mins)
[x] Collect Step 3 (Aspirate out all liquid. Only pellet should remain. Store sample in lab box in freezer)
[x] Change media
[x] Prep self for PCR tomorrow with previous collection
20x & 40x on microscope didn't seem proper. Found Chris (lab tech) to ask in order to verify. Chris confirmed microscope was not functioning properly and adjusted it until it could take photos in live view again. Told Chris what I noticed about the microscope back in Dec. and tried to tell people about (fluorescent function seemed on even if it was off, concerned it affected the quality/consistency of my cell photos). After taking all the photos @4x, 10x, 20x, & 40x in black & white, I double checked to make sure the photos saved to my thumb drive.
The photos did not save, so I re-took photos again in order to manually save at each magnification.
Cell count data:
[EDIT JUNE 29TH, 2016 : TAKING OUT THE RESULTS FROM THIS BLOG UNTIL I HAVE MY THESIS ACCEPTED AND PUBLISHED. LAB DIARY WILL BE MOSTLY DOCUMENTING DAILY LABLIFE, PROTOCOLS, + TROUBLESHOOTING FROM HERE ON OUT.]
The following data are what each collections are starting at:
To convert concentration to number of cells, multiply by volume:
Resuspended in 3mL to count, therefore, multiply Total concentration by 3.
The following are cell photos from today: