Week 6 - October 21, 2022

 Tuesday, October 18, 2022 

Making TSA: 

  • Did measurements from 1 Liter to 500 mL 

    • 20 g of Tryptic Soy Agar 

    • For the slants added 1 g of agar 

  • Placed 20 g of TSA into a beaker containing 100 ml of D.I. Water 

  • Put beaker onto hot plate and stirred it 

  • Wanted it to be hot and molten but never boiling 

    • Wanted to keep it spinning 

    • Made sure everything was dissolved completely before removing from the hot plate

  • Once it was completely dissolved, we removed it from the hot plate and began to pipette 

    • 6 ml into each tube 

    • If it began to solidify, switch out pipette and put the TSA back onto the hot plate, spin until it is a liquid again and can continue to be used  

  • Placed lids onto each test tube 

    • Test tubes were then autoclaved 


Friday, October 21, 2022 

Plating: 

On Friday, my partners and I learned how to plate. We used the gels that we had made last Friday October 14, 2022. The gels contained LB Broth and the antibiotic Ampicillin. To plate, we used a freeze bac which was E.coli that had been cryoprotected. I was able to work under the hood for the first time. 

Working Under the Hood:

  • Turn on the UV light for 15 minutes prior to beginning 

    • This will sterilize the hood in order to be able to work there

  • After the 15 minutes, turn on the light 

  • Wipe down all equipment with ethanol and kim wipes

  • Lift up the glass panel of the fume hood 

  • Turn the blower on 

Plating: 

  • Decide which side of the ring to use 

    • Smaller loop or larger loop 

  • Open the sterilized loop from the opposite side of the loop you want to use

    • If you want to use the smaller loop, open it from the larger side

    • If you want to use the larger loop, open it from the smaller side

  • Dip the loop into the tube of E.coli (freeze bac) 

    • You want to dip right into the top of the liquid 

    • You don’t need to get any of the frozen sludge 

  • Streak onto the plate 

  • When you are done with the streak, cover the freeze bac with its lid and cover plate with its lid

    • Make sure the plates have the smaller part of the plate facing up (lid down) 

  • Incubate at 22°C 

 

Comments

Post a Comment

Popular posts from this blog

Spring 2024: Week 14 - April 29, 2024 - May 3, 2024

Image
  Introduction  Last week we attempted Gibson Assembly for the first time. As we interpreted the results closely, we seem to have been successful. We had a faint band at around 2,500 bp and our fragment was 2,527 bp. Since the band was faint, we are going to attempt to amplify it this week. Methods 4/29/24 We kept our broth culture of D. metalli in the fridge and the biofilm has continued to form. Since it is one of the few species in the Deinococcus genus that forms a biofilm, we were interested in seeing it under the microscope. We decided to do a gram stain on it.  4/30/24 In order to amplify our Gibson product, we decided to run it through PCR again. This time, we did Q5 PCR to get a higher yield than what we previously got. We ran two different samples. The first sample was 48 µL of mastermix + 2 µL of our 3-way Gibson that had been put through PCR last week. The second tube was the original Gibson product that had not been put through PCR.  At the sam...
Read more

Spring 2024 Week 2: January 29, 2024 - February 2, 2024

Image
  Introduction:  This week we continued our work with E. coli pRHAM. Last friday, we completed a plasmid extraction in order to get the pRHAM plasmid out. This week, we focused on confirming that we successfully extracted the plasmid before moving onto using that sample in PCR. If both of these go smoothly, we will be able to begin assembly, using Gibson Assembly, at the beginning of next week.  Methods: 1/29/2024  Using gel electrophoresis was our way to confirm if we had extracted the pRHAM plasmid. We mixed 2 μL of UV blue dye with 10 μL of our plasmid sample in an Eppendorf tube. We spun our tubes down in the pop spinner to make sure it was all one homogenous liquid. Since the plasmid’s size is 947 kb, we added a 1 kb ladder into one of the wells so that we could compare the size of our band to the ladder. We let the gel run and checked it under the UV light.   1/30/2024 Using our plasmid sample, we ran a short-amp PCR. We mixed 8 μL of mastermix ...
Read more

Spring 2024: Week 15 - May 6, 2024 - May 10, 2024

Image
  Introduction  During our last week in the lab, we attempted to perform Gibson Assembly with the 3-way fragment we previously assembled and the vector (pRAD1 plasmid). We are also preparing for the summer semester when we will also continue our work with D. metalli .  Methods  5/6/24  We are running out of pRAD1 plasmid extractions. We will need to extract more since we will continue to use it for this project and also for our D. metalli project. We inoculated E. coli pRAD1 into a flask of 25 ml of LB broth. We added ampicillin into the broth before inoculating. Since we plan on extracting within 24 hrs, we placed the flask in the shaking incubator overnight.  We also performed Gibson Assembly. This time we are attempting to assemble the 3-way fragment and the pRAD1 vector. If it assembles correctly, we will have a fragment that is 3,500 kb. We put the 3-way fragment and the vector in a PCR tube with the Gibson mastermix. The tube was incubated at 50℃ f...
Read more