Week 14: Making new samples and inoculations

 Introduction: 

Due to the contamination of our D.Aquaticus sample in week 13, I conducted new inoculated samples this week. Instead of just one sample, I conducted with 3 samples to ensure extra security and support. There was a lot of variables that went into the contamination of our D. Aquaticus sample so this time I wanted to do multiple samples to avoid situations like that. It will also give me extra samples to return back on (if they are not contaminated) rather than having to retry the inoculations again. It will also save on materials and ingredients. 

Process: 

I gathered 3 flasks with each one being A, B, and C. These will all have possibly the same amount of bacteria (one sterilized loopful) and TGY (25 mL). 

First I pipetted 25mL of clean TGY into each of my flasks and then autoclave it to ensure that all of them are completely clean of any cells. After that I waited for them to cool down (almost to room temperature) to avoid killing D. Aquaticus cells when inoculating. 

Fig. 1 25mL of autoclaved TGY 


Next, I conducted inoculation by heating up and sterilizing the transferring loop. I also transferred our D. Aquaticus from the 11/10 sample into each of the flasks; sterilizing the loop each time I transfer. 

Then, I put the three samples in the incubator and will check back next week for any growth and will take OD samples of each. 


Comments

Post a Comment

Popular posts from this blog

Week 15 (4/15-4/19): Attempting confirmation cells on our existing transformed cells

Image
  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 ...
Read more

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

Image
  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   ...
Read more

2/12-2/18: Week 6 Tetracycline vs Caeni efflux pump experiment

Image
 Introduction In lab this week, my group and I did one more testing with Tetracycline and Caeni. However, based on the results last week, we decided to do the concentrations below 1 ug/ml. The concentrations are 0 ug/ml, 0.25 ug/ml, 0.5 ug/ml, and 0.75 ug/ml. We expect at least some growth between 0 ug/ml and 0.5 ug/ml.  Procedure  To prep, we made four 25ml flasks of normal TGY agar. The reason for this is because we want to ensure that each TGY plates we will be pouring is evenly 25ml. We then recieved our 2.5 ug/ml tetracycline stock from Chad. We also grew our caeni in a 10ml TGY flask.  To keep in mind, our OD value of caeni was 2.44 after 3-4 days of growth and was diluted to 0.97. 1. We label each of four 25ml flasks of TGY agar their desired tetracycline concentrations and also labeled four empty plates the corresponding concentrations to the flasks.                    Flask #1 and Plate #1: 0ug/ml...
Read more