Deja Vu Birney's TRAIN Blog

     This week in lab, we began with starting over with our experiment. Last week, our RNA results came out very low and dirty, most likely due to improper handling of samples. For this experiment, we are looking to see if the gene PFS in Deinococcus radiodurans is expressing when put under oxidative stress with hydrogen peroxide.       We started growing our samples on Monday for a total of 24 hours of growth. Tuesday, we started the dilution process. We ensured all 6 samples were individually measured for 1 OD600. After diluting our samples, we the introduced 3 of our 200uL of  D. rad.  in TGY samples to 200 uL of 200mM of H2O2 for 30 minutes. Realistically the H2O2 was at 100mM due to it being diluted once it was added to the samples. For our controls, we just added 200uL of TGY for consistency in volume. Once incubation was completed, the cells for all the samples were washed thoroughly with TGY and pelleted for RNA isolation.  ...

     This week in lab, we continued to isolate RNA from D. rad. The difference is we are actually starting the experiment part of our project. Last week we focus on verifying our primers for our reference genes Sec A and GAP 3. It was determined that because Sec A was 105% efficient and GAP 3 was 97% efficient, we were ready to move on with our experiment. The gene that we are targeting is PFS. PFS gene is a gene that encodes for methylthioadenosine/S-adenosylhomocysteine nucleosidase. Its important to the activated methyl cycle. So we are trying to see if oxidative stress to this gene, effects the AMC in any way.      Our first step was to grow cells. We are continuing to grow D. rad for 24 hours but we changed the volume of TGY we are growing it in to 10mL. After 24 hours of growth, we determine that we wanted to do three controls and three tests with a total volume of 200uL of cells at 1.0 OD600. We diluted it by tube and ended up with the following resu...

                This week in lab, we did RNA isolation on   deinococcus radiodurans.   The reason for doing this is so we could synthesize cDNA. cDNA is a template we are using to study gene expression in   D. rad  when it’s exposed to oxidative stress.   For now, we are doing qPCR for our primers on the gene Sec A to see if it’s a good reference for our experiment.               On Monday,  D. rad  was inoculated into 250mL of TGY media as well as three TGY agar plates. The broth is being used for RNA isolation and the plates for freeze back. On Tuesday, a gram stain was performed on the inoculated TGY media and determined that there was no contamination and RNA isolation was ready to be performed. 3mL of our sample was spun down into a 1mL Eppendorf tube. The process was performed as such:   D. rad  inoculat...

     At the start of this semester, we started with doing a "Biotech Bootcamp." This week of instruction included lab safety, lab protocol, as well as having time to teach/remind students how to pipette and make media/agar. One important protocol we talked about was regarding labeling. We clarified that all media, agar, bacteria, ect. needs to be labeled with the date, contents, initials, volume, concentration, type of bacteria (this is all depending on what the contents are). Labeling also was an issue when it came to waste beakers in the lab. Now all beakers that contain waste must be labeled clarifying whether it was chemical or bacterial waste. We also discussed lab cleanliness. This included washing your hands before and after doing anything in the lab and cleaning off surfaces. Something that was an issue last semester was people leaving powdered media on the scale after measuring, and not wiping things down property. All equipment must be cleaned before and af...

       These past couple weeks in lab have been very interesting. First, we decided to give the right fragment of   deinococcus radiodurans   and tetracycline from   E. coli  a try. We are doing this because we haven’t seen much success in adding our right fragment to our left and tet fragments. We initially had isolated more right fragment via gel excision. We had performed two different right isolations. Our ng/μL reading on R 1   isolation was 18.8 ng/μL, and for our R 2   isolation it was 27.7ng/μL. The sample we chose for our overlap reaction was R 2 . We then decided to do an 8-sample overlap heat gradient of right fragment and tetracycline from 35 °C to 60°C. The recipe we used is below:   11μL 7μL 2μL 4μL  Master Mix PCR H 2 0 Right Fragment Tetracycline Fragment             From L to R: 60°C, 57.9°C, 54.6°C, 50.1°C, 44.5°C, 39.6°C, 36.7°C, 35°C   The two best bands we saw were at 57°C...

       Last week in lab we had run our left fragment from   Deinococcus radiodurans,   and our tetracycline fragment from   E. coli.  On a heat gradient from 34 ° C to 54 ° C. This gradient led to our discovery of the ideal temperature for a fragment overlap, that being 54 ° C. With this, we decided to play around and do another gradient. This gradient was from 52 ° C to 70 ° C with a total of 8 reactions. We did have to make more left and tetracycline fragments as we were running low, so we had spent most of our time amplifying those samples and gel excising them. For our overlap reactions, we used the recipe below:   5µL 7µL 11µL 1µL Left Tet Master Mix PCR H 2 O   We ran these 8 samples through PCR in their gradient. We then amplified the samples using the recipe below:   5µL 10.75µL 2.25µL 2µL Overlap Sample Master Mix PCR H 2 O Primer Mix  (A & D)   We then run these 8 samples on a big 1.2% gel for about an hour. ...

This week, we did a PCR overlap heat gradient ranging from 34°C to 54°C on our left fragment of  deinococcus radiodurans  and our tetracyline fragment from  E. coli.  When we ran this reaction on a gel, there was absolutely no DNA showing up. We assumed there was maybe a problem with the pipetting of the fragments into the tubes when they were going through PCR for amplification, and we decided that it would be best to start from the beginning again to ensure that there was no error. We then proceeded to redo the overlap as well as the amplification process at 34°C to 54°C. For these reactions, we made sure they were equal molar. We first had to dilute our samples down to left being 10ng/µL and tet being around 5ng/µL. We did this by getting our original samples and adding PCR water. With our goal of having a 50ng/µL total for left and a 35ng/µL for tet, we loaded the amount below.   5µL 7µL 11µL 1µL Left Tet Master Mix PCR H 2 O   With this ...