S-STEM Scholar Alex Hugenberg-Cox Blog

This week we were finally able to do qPCR. We didn't have lab Monday, so we started Tuesday with diluting our samples to prep for RNA isolation. We tried to get as close as possible to an OD600 of 1. After a bit of work, we ended up with the closest dilutions prier to RNA isolation that we've had so far.  Before Dilutions: T1  2.86 T2  2.14 T3  2.73 C1  3.06 C2  3.20 C3  3.11   After Dilutions: T1  0.99 T2  1.01 T3  0.99 C1  0.98 C2  1.01 C3  0.99 Wednesday we did RNA isolation and cDNA synthesis. After RNA our samples we nanodropped the samples to see our concentration.         ng/ μl       A260/280   A260/230 T1  6.6         1.83            1.08 T2  16.3       1.97            1.08 T3  32.6       2.06            1.35 C1  35.3       1.96            1.09 C2  24.2       2.00            1.20 C3  23.9       1.89            0.81 After that we did cDNA synthesis. We did our best to load 92ng of RNA for each sample. T1 14uL - 92.4ng T2 5.7uL - 92.9ng T3 2.8uL - 91.28ng C1 2.6uL - 91.7

We started Monday by doing a RNA isolation. Before we could begin the actual RNA Isolation procedures, we needed to first dilute our samples (close to 1.00 ng/μl) and expose the test cells to Hydrogen Peroxide. Post Dilution Values- T1 1.04 A600 T2 1.04 A600 T3 1.07 A600 C1 1.04 A600 C2 1.04 A600 C3 1.04 A600 After Dilution, we exposed T1, T2 and T3 to Hydrogen Peroxide. We had 200 μl of sample, and added 200 μl of H2O2 (200 mM) for the 1:1 ratio of sample to H2O2 needed. We also added 200 μl of media to C1, C2 and C3 in order to keep the volume consistent. After adding either H2O2 or Media to all of the samles, we incubated at 30 °C for 30 minutes. After  thoroughly cleaning the inside of the hood, and all the equipment needed,  we moved on to the actual RNA Isolation Procedures.  RNA Isolation Procedures- We start by centrifuging and resuspending the cell pellet in 800 μl of RNA Lysis buffer and transfer the mixture into a glass bead beating tube. We then put the tubes into a bead be

     Leading up to the cDNA synthesis other members of the group made and inoculated plates/broth, graham stained and performed RNA isolation and converted RNA to cDNA.  Image of D.rad Graham Stain Picture taken through microscope of  small pink circular cells     For cDNA, we start with our RNA. After nanodropping the RNA to ensure the concentrations are good enough to perform cDNA synthesis on, we could run a gel to verify the quality of the sample. We than serial dilute the RNA to get our ideal dilution (from previous experiments around 1/1,000 and 1:10,000)      We take 1 ng of RNA and combine it Nucleotide free water in order to read 14  μl total volume. We than combine the 14 μl with 2 μl DNase master mix (.5 μl DNase and 1.5 μl DNase buffer). We than use the PCR machine to incubate the reaction.  DNA Digestion 25 °C for 5 min. DNase inactivation 75 °C for 5 min. than held at 4 °C     After the reaction we add 4  μl of reverse transcription master mix and incubate the reaction ag

     This semester we began lab with a 'biotech boot camp' to both remind returning students and tech new ones about different procedures and protocols for the lab. We covered topics like metric units and conversions, lab notebook etiquette and labeling things. We also went over creating media, using pipettes, experimental design, serial dilution and some basic lab math.      At the end of boot camp we went over our new projects, as this semester we will be focusing on  Deinococcus  papers. I will be working on a project lead by Chad; AMC pathways and qPCR. We went over the basic pathway of the project, a lot of vocabulary and read a few papers covering different techniques we will be covering during our project.            We will putting D.rad under oxidative stress using Hydrogen peroxide. This will cause the cells to start distribute and receive 'warning signals' called AHL's. 30 minutes after being put in hydrogen peroxide, the cells start to produce enzymes th