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Plasmid Extraction

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    This week we started with running a couple gels for our different fragments. I worked on the left fragment, which unfortunately did not give the results we were hoping for. Since it was all the same sample, just ran on different temperatures in the thermal-cycler, we combined them all and will work later on cleaning them up.     We took our e.coli broth and used that as our base. Originally we started with only 1mL of our sample, and after the plasmid extraction was completed, there was very little DNA in the sample. We then used 5mL to extract enough DNA to work with. We started with centrifuging 1mL at a time and discarding the supernate. After we did 5 different cycles of centrifuging we had our pellet     Once we had the pellet we resuspended it with 200 micro-liters of our B1 buffer. Then added an additional 200 micro-liters of the B2 buffer (Lysis Buffer), followed by gently inverting the sample until it was mixed. An additional 400 micro-lit...
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PCR

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 Introduction     This week we hit the ground running. We started by taking our fragments of DNA and started to put them together. We took 3 different samples, two being DNA and the other being our plasmid. Inside of the samples we had 1 micro-liter of our forward primer, 1 micro-liter of our reverse primer, 2 micro-liters of the DNA/Plasmid, and finally 16 micro-liters of master mix. Once all the samples were combined, they were placed inside the thermal cycler to run their course. Upon completion of the thermal-cycler, we came to find out there was a mix-up in which DNA/Plasmid was placed into our tetramycin sample. Since we took the time to create the samples and had them run in the thermal cycler, we decided we would run the sample on the nano-drop as well as run a gel.      As seen from the gel there are a couple ghost fragments as well as blurriness, we'll be working towards cleaning the sample for the cleanest fragments we can get.    ...
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Week 1

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 Introduction     Thursday was spent preparing bacteria for whenever the supplies we need arrive. We started by dyeing some samples with Crystal Violet in hopes of it sticking to biomass. After the samples had grown for 48 hours, we evacuated the walls (emptying the trays) and then rinsed them with water, then dumped them out again. After the samples had been successfully evacuated, it was time to apply the dye. We placed 225 micro-liters of Crystal Violet into the samples, allowed them to sit at room temperature for 20 minutes, then repeated the evacuation steps. After the dish has been successfully evacuated, we allow the samples to rest for 24 hours at room temperature being placed at an angle to dry.      Come Tuesday, our samples will hopefully reflect the growth of biomass, and we will continue the process of inserting a new fragment into our sample's DNA. 
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Restriction Enzyme

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  Introduction           We started this week off with restriction enzymes. Learning about restriction enzymes is imperative to developing laboratory methods as well as learning about genetic engineering. A restricted enzyme is a protein that has been isolated from bacteria, producing DNA fragments. We started with 2 micro-liters of restricted enzymes, 10 micro-liters of our buffer, 8 micro-liters of our kanamycin DNA, and then 80 micro-liters of PCR water. The main goal was to remove pst1 from our sample's DNA.  After we had our samples combined, we incubated at 37 degrees celsius for 15 minutes, which was then followed by inactive at 80 degrees celsius for 20 minutes. We then loaded the samples into our gels and then ran the gels at 85 mA for around 30 minutes.  Once the gel had run its course, we placed the gel over the UV light and found out the kanamycin did not split how we were hoping it would.  The following day we ran an additio...
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Gel Extraction

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  Introduction     In the process of learning new things, sometimes science can become redundant. This week we worked on the Left Flank of our sample and went through the gel extraction process. We started with making a 1.5% gel to run our samples through. To make this we need 30 Ml of 1X TAE and 450 mg of agarose powder. While we waited for our gels to solidify, we tested our samples on the nano-drop.  From this we got:   Ng/Micro-Liters A260/A280 A260/A230 L1 245.9 2.01 2.04 L2 208.4 2.01 1.39 L3 233.9 2.03 2.07 L4 209.0 2.04 2.09     After the gels finished solidifying, we added our samples. We ran 18 micro-liters of the DNA sample, and 2 micro-liters of our blue dye.      After the gels had finished running their course, we placed them ...
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Nano-Drop

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 Introduction     This week was preparation leading into next week's project, ethanol precipitation. Ethanol precipitation is used for concentrating and de-salting nucleic acid (DNA or RNA). This week we were running our samples through gels and then extracting our samples once they've run through the gels. We started with a different variation of our usual 1% gel, this time we used a 1.5% gel. For this, we needed 30 mL of 1 time TAE and 450 mg of agarose powder. With the addition of an extra 5 seconds in the microwave and 150 mg of agarose powder, our gel is made. While we were running our gels,      We set the Nano-Drop to Double Stran DNA and tested the samples.  The reason there are 2 pictures is that I ended the experiment by accident....     By the time we finished on the Nano-Drop our gels had finished and it was time to place them over the UV light and extract the sample.      We measured each 2mL tube that the samples ...
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Alcohol Precipitation

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 Introduction      Last week we worked with the Left Flank, as well as the Kanamycin resistant gene from e.coli, in our effort to transform Deinococcus Radiodurans. This week we worked with the Right Flank. First, we started with making our 1% gel to run the samples through. While we waited for the gel to solidify, we Nano-Dropped our Right Flank sample.      From this, we got: 280 -2.71, 260 -5.10, 230 -2.27      Finally, our gel had become as solid as we wanted. We loaded our 20 micro-liter sample (2 micro-liters of dye, 18 micro-liters of DNA) into 2 wells, and made sure to load the ladder. After we ran the gel it was time to place it over UV light and carefully remove the fragment.  This photo is from last week but added to emphasize the procedure.      After we extracted the fragments we placed the Right Flank as well as our other fragments into the -20 degrees Celsius freezer to chill overnight. The following mo...
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