October 14th-October 18th

 Week Four (Oct 14-18)

We started the week by performing a plasmid extraction that we still need to confirm with a gel.


Additionally, we also still did not reach a conclusion as to what happened revolving the previous gel we had done on our pgK. We think that the wrong ladder was used which caused everything to go wrong since we don’t know what ladder it is, which is why it’s so essential to write and label everything down.


Afterwards, we went over lab math, how to pipette, how to load and run a gel, and doing a digest.


SOP Loading a Ladder

  • Start with largest volume

  • In this case, we used 5 ul sample, 5 ul water, and 2 ul of dye

  • We started with our 5 ul water for sample and 4ul into our ladder.

  • Afterwards, we added our dye. When adding dye, submerge your pipette tip into the water so that the water is able to ‘grab’ onto the dye.

  • Then, add the DNA. No DNA goes into the ladder since that’s what tells us the base pairs we are seeing.

  • Pour TAE over the wells first.

  • For this gel, we ran it at 50 amps.


SOP Gram Stain

Pink = Neg

Purple = Pos


Procedure

  • Start by spraying the slide with ethanol and wipe

  • Then proceed to light the bunsen burner and heat up wire until orange in order to kill bacteria.

  • Open the plate just a little and drag your loop through the top of agar once it has been given time to cool. Then, swipe on slide

  • Proceed by sliding over flame till dry


For actual gram stain

  1. Add drop of crystal violet onto slide for only one minute. The crystal violet stains the peptoglycin purple.

  2. Spray with D.I. Water, but not the actual circle. Keep spray above where the crayon is so it washes down.

  3. Proceed by adding 1-2 drops of iodine for 1 minute.

  4. Repeat #2

  5. Add drop of ethanol for around 5-10 seconds, 15 if truly needed. 

  6. Repeat #2

  7. Add 1-2 drops of saffronin for 45 seconds to 1 minute.

  8. Repeat #2

  9. Blot to dry and proceed to microscope


SOP Inoculation

  • Start by washing hands up to elbows and clean the space you are working at

  • Turn on the gas and light the bunsen burner

  • Place the loop over flame until the entire wire is orange, then wait 30 seconds

  • Before committing to your swipe, test agar and then do a streak. For this time, I did a one way streak, but a four way streak is preferred since it’s more efficient but also more difficult for some.

  • It’s essential to move very fast to avoid contamination

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