S-STEM Scholar Anh Nguyen's Blog

  Introduction     In lab this week, we observe our PW1 Cas9 transformation results from Week 12. To recap, we plated P1 C and P1. C (reloaded 600 ul of culture after first spin), P2 LBR and P2. LBR (adding LB broth and reverting), and P3 LB (only adding LB broth). We also redid transformation of both pRAD 1 and PW1 Cas9 again. Below is our nano-dropping and cell density of each reaction.  Table  - pRAD 1  Reactions     ng/ul     A260/A280     A260/A230     Amt of Elusion Buffer Added P1                     83.8          1.86                      1.65                         10 ul  P2                    38.1          1...

  Introduction      After our transformation before spring break, we want to venture out into performing transformation with different plasmids in E.coli . One notable plasmid that we worked on this week was PW1 Cas9. At the same time, we are still retrying to pRad 1 but this time, during the extraction process, we will test with different amount of elusion buffer since we want more mass densities in our cells. The less elusion buffer we use, the more dense the cells are going to be. pRad 1 Procedure and Results     For extracting pRad 1, we plan to do three different amounts of elusion buffer: 10 ul, 20 ul, and 30 ul of elusion buffer. We will do 6 reactions, 3 being set #1 (10 ul, 20 ul, and 30 ul of elusion) and the other 3 being duplicates. Here are the results taken from the nanodrop:                    ug/ml     A260/A280     A260/A230 E1 (10ul)     17.5  ...

  Introduction     This week in lab was busy and productive as we were to complete our transformation outline before spring break. In the beginning of the week, we loaded our extracted plasmid that we did in week 9 onto a gel and run it. At the same time, our d.caeni successfully grew in broth without any contamination so we were able to do competent cells. After running the gel and competent cells, we were able to perform the transformation procedure.  Gel Procedure     To load a gel, we must obtain the required materials: 6 ul of loading dye and 6 ul of the 1k base pair ladder (we know that pRad 1 has a 7kb ladder so we want to expect that on the gel). To make the gel we mixed .30g of GelRed agarose and 30ml of TAE.      After obtaining these materials, we began to mix together the solutions. Since we want to run 3 lanes we would required 5 ul of pRAD1, 5 ul of PCR water, 2 ul of loading dye and 6 ul of the ladder.  Gel Results   ...

  Introduction      We begin our experiment this week by preparing the correct media to grow both of our E.coli and D.caeni.  Luckily, E.coli only takes a day to grow on both plates and broth so we were able to get started on our plasmid extraction earlier in the week. While we are running our extraction, we are also growing our D.caeni in plates and broth since it was known that the cells grew slower. Below are the steps we took to run our plasmid extraction to get pRad 1 from E.coli cells.  Procedure Before we perform plasmid extraction, we had to OD value our E.coli broth sample and gram-stain the cells to find out if the samples are fresh and clean. We are also going to run 4 reactions. To do this, we would need 8 ephindorph tubes (4 for set #1 and 4 for set #2), 4 collection tubes, waste beaker, and the Zyppy Plasmid Kit.  1. Pipette 600 ul of grown E.coli broth into each ephindorph tubes (set #1)     ->  Centrifuge tubes for 1 m...

  Introduction     As our project with MIC comes to an end, my partner and I will transition towards transformation of d.caeni with pRad1.  Unlike MIC, transformation is a lot denser and more complicated since it is regarded as a parasexual process that involves two partners: exogenous DNA and a recipient cell. For this transformation, we will be working with pRad 1 as our exogenous DNA and d.caeni as our recipient cell.      To get ahold of pRad 1, we must extract it from E.coli which is known to have the pRad 1 plasmid inside. Below is our map and outline of the transformation.  Procedure 1. Obtain E.coli sample from freezebacks (we want fresh new samples)  2. Streak and grow E.coli in LB with ampicillin plates (the stock concentration of amp: 25 ug/ml) 3. Grow E.coli in LB with ampicillin broth (the stock concentration of amp: 25 ug/ml)      ----> The reason to why E.coli must be grown in LB and amp is because since E.col...