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Week 14 (4/8-4/13): Working on scientific poster and passaging transformed cells

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  Introduction In lab this week, we did not perform any experiments because we mainly focused on editing and writing our scientific poster about our transformation project with E.coli plasmids and D. caeni  for our conference on 4/13.  The Poster In the poster, we talked about the purpose of this experiment and we discussed about what Deinococcus species was successfully transformed in the past that allowed us to continuing working with E.coli plasmids and the Deinococcus species. In previous experiments, Deinoccocus radiodurans was proven to be successfully transformed with pRAD1 . Below shows the results of the transformed cells on TGY plates with 3 ug/ml chloramphenicol.  These plates show 1 ug of pRAD1.  Below is another result of the transformed cells in a gel performed from colony PCR as confirmation.  Lane 1: 100 BP Ladder Lane 3-5: 861 BP of PCR product Since we have confirmation and evidence that a Deinococcus species have the ability to be transfo...
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Week 16 (4/22-4/26): Regrowing our transformed cells and PCR Gel

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  Introduction  In Week 16, we grew broth culture and performed PCR on our transformed cells which we loaded onto a gel this week. The issue we had about our culture was that there was little to no growth from our transformed cells in antibiotics. This time we will regrow them again in broth but also plates as it may give us different results due to different underlying factors.  Results Below is our PCR Gel.  This was loaded with a 100 BP ladder. In each wells contains 5 ul of our 783 ng PCR solution with 2 ul of dye. From there picture shown, there is not much of the genetic appearance. We believe that using only 5 ul of PCR solution wasn't sufficient enough and will plan to use 10 ul of it in the future.  For regrowing our transformed cells, we passaged 135ng pwtCas9, 550ng pwtCas9, 783ng pwtCas9, and 803ng pwtCas9 and control D. caeni  plates onto new TGY plates with the same antibiotic concentration. At the same time, we also grew them into new centrif...
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Week 15 (4/15-4/19): Attempting confirmation cells on our existing transformed cells

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  Introduction Unfortunately since we could not successfully transformed pwtCas9 with D. caeni in Week 13, we will be using existing transformed cells that we have successfully gotten in the other previous experiments to attempt confirmation cells. There are two ways we can perform confirmation; PCR and Plasmid Prep. PCR is essentially the reading of genomic sequences of the cells and since we know that these transformed cells were successfully resistant to tetracycline, we want to see the resistant sequence gene once we load the PCR into a gel. Similarly, plasmid prep allows us to perform plasmid extraction on our transformed cell and loading them into a gel to see if it has the pwtCas9 plasmid within the cells.  Procedure For this confirmation, we used 4 different concentrations of the transformed cells and two controls: - 135 ng pwtCas9  - 550 ng pwtCas9  - 783 ng pwtCas9  - 803 ng pwtCas9  - (+) D. caeni (no antibiotics) - (-) D. caeni (with 2 ug/ml of ...
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Week 13 (4/1-4/5): Repeating transformation with pwTCas9 plasmid and D.caeni

Introduction In lab this week, we repeated a transformation protocol with the pwtCas9 plasmid and D. caeni  hoping that it can give us good results on plates to do plasmid confirmation. The beginning of the week comprise of growing plasmid culture in 20 mL of LB broth with 50 ug/ml of ampicillin. We also prepared TGY agar plates with 2ug/ml of tetracycline to observe our transformed cell growth. We hope that there will be transformed cells grown on the plates as the pwtCas9 plasmid was proven to be resistant to tetracycline.  Plasmid Extraction Procedure Below is a list comprising of how we performed plasmid extractions with pwtCas9.  Solutions         # of Cell Packing          Resuspension          Elusion Buffer Amt PC1                              1                ...
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Week 12 3/25-3/31: Transformation of PW1 Cas 9 results and more transformation

  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...
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Week 11 3/18-3/24: Trying transformation with a different plasmid

  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  ...
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Week 9 3/4-3/10: Loading into gel, do competent cells, transformation, and repeat plasmid extractions

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  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   ...
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