week of 9/9 to 9/13

During this week I was shadowing my current lab group: 
  • Cut Kanamycin from its X-bai site and ligate it to PGRNA. 
  • learned that Sonora dionecoccus is a bacteria that is highly resistant to UV light. 
  • gram-stained Cas-9, however, they looked contaminated. 
  • learned that competent cells are cells that can easily take in foreign DNA, making them good for transformation.
  • Performed a transformation of Deinococcus radiodurans
    • Thaw competent cells. 
    • Aliquot 100 µL of competent cells into a microcentrifuge tube. 
    • Add 1 µL of plasmid solution to a microcentrifuge tube with competent cells. 
    • place on ice for 15 mins
    • Place in incubator at 37°C for 45 mins.
    • agitate the tube every 15 minutes 
    • Transfer contents into a 15 ml centrifuge tube with 4 ml of TGY broth. 
    • Incubate in a shaking incubator for 16 hours (overnight). 
    • after 16 hours, plate onto R2A agar plates with antiobiotic for 4 day
  • Made TGY agar plates with ampicillin for D. Sonora. 
  • materials for TGY Media
    • DI Water 
    • Erlenmeyer flask 
    • graduated cylinder 
    • aluminum foil 
    • scale
    • spatula 
    • tryptone
    • yeast
    • dextrose
    • 0.5% agar granulated 
  • passaging Sonora on 1 0.2 ampicillin plate and 1 regular TGY plate and after passaging place in 30C incubator
  • How to make a 1% agar gel
    • 30 ml of TAE Buffer
    • 0.03 g of gel red agarose 
    • mix in a flask double the amount needed
    • microwave for 1 minute 
    • let cool for 5 minutes, then pour 

Comments

Post a Comment

Popular posts from this blog

week 10/28- 11/1

Plasmid extraction of Pd-Cas9  O.D. value is A: 5.35, B: 5.44 Only 2 uLs of ellusion buffer were used for a high concentration because it will only be needed for transformation.  Nanodop reading of PdCAS-9 E. coli  42.4 ng/µL concentrations of tube A (A260/A280: 1.84) (A260/A230: 2.99)  Tube B: 36.4 ng/µL (A260/A280: 1.85) (A260/A230: 1.66)  For transformation of Pd Cas9, only 1000 ng is needed and at least 10 µL of volume Gram stain of pd cas-9 E. coli  Contaminated so gram-stained parent plate to check if the contamination is coming from the source.  A tube of Pd cas 9 was found in the -20 freezer so passaged that on plates and created a freezback. single digested pgK with xbar, inoculated our TGY and LB plates ligation of PGRNA and kanamycin (5:1 ratio) If ligation is not successful, Lambda DNA will be used as a control to figure out exactly what we are doing wrong in the process. 
Read more

week 10/14 -19

We started the week by doing a plasmid extraction. We still need to confirm the results with a gel. pgK Gel. During these next few weeks, plasmid extractions have all been coming out with bad nanodrop results, which left us on a standstill and confused as to what could possibly be going wrong with the extractions.  still haven’t figured out what went wrong with the previous gel for pgK. Might have used the wrong ladder, which threw everything off. This highlights how important it is to label everything properly and write things down.
Read more

week 10/21-25

This week was mostly about gram-staining pd cas9 and learning to use the 4-way streak method for better colonies of growth on the agar plates. I learned it was best practice to streak the plate facing down for less risk of contamination, but I prefer to streak face up for more accuracy.      When gram-staining growth from these plates, in this case Pd Cas9, it's good to know that a little more time on the ethanol step won't harm the quality of the stain too much, especially if the cells are compacted.  After these plates were gramstained, they were passaged onto R2A and TGY plates poured during the week. 
Read more