Gram Staining, RNA Isolation, and Inoculation

 February 4, 2024

Introduction

This week included RNA extraction from E. coli with the intention of working towards qPCR, but also to test the kit for organic contaminants. Also this week was an introduction into gram staining, which is used specifically to verify samples and to check for contamination. Finally this week was inoculation, the process of implanting a microbe or other organism onto a plate for further testing later. 

Methods

For RNA extraction and purification, the steps are as follows: resuspend fresh or frozen cell pellet in 800uL RNA Lysis buffer and transfer the mixture into ZR BashingBead Lysis Tube. Secure the tube in a bead beater fitted with 2mL tube hold assembly and process. Centrifuge the tubes for 1 minute to pellet debris, then let sit in an ice bath for 2 minutes. Repeat ten times, then transfer up to 400uL of cleared supernatant into a zymospin IIICG column in a collection tube and centrifuge, saving the flow-through. Add an equal volume ethanol (95-100%) and mix well. Transfer into a zymospin IICR column and centrifuge 1 minute, discarding the flow-through. Add 400uL RNA prep buffer to the column, centrifuge 1 minute, discard flow-through. Add 700uL RNA wash buffer, centrifuge 1 minute, discard flow-through. Add 400uL RNA wash buffer and centrifuge 1 minute to ensure complete removal of the wash buffer, then transfer the column into a nuclease-free tube. Finally, add 50 uL of DNase/Rnase free water directly to the column matrix and centrifuge. The process for gram staining is as follows: clean station and gather materials. Clean slide with ethanol by applying ethanol to a kimwipe, then wiping the slide. Draw a circle with a wax pencil directly onto the slide, number the slide if multiple. Heat inoculation loop in flame until glowing then allow to cool. To collect sample from beaker, wave beaker over flame to prevent contamination the use loop to collect and recap sample. Smear collected sample in the circle, then heat fix by passing the slide over the flame briefly. Bring slide over to a sink, then flood plane with crystal violent and let sit for 1 minute before flushing with DI water. Next flood the plane with Grams iodine and let sit for 1 minute, then flush with DI water, then waterfall with ethanol before flushing with DI water again. Once again fill the plane with safranin, let sit for 50 seconds and then flush with DI water. Press slide dry with bibulous paper and then examine sample with microscope. If the sample is purple, it is gram positive. If it’s pink, it’s gram negative. Finally I was shown a demonstration of inoculation. First clean the workbench with ethanol, then obtain plates for streaking. Cure the inoculation loop in flame and then allow to cool before picking up a culture from the old plate and spread onto the new plate, going corner to corner before dragging into the center. Repeat on a second plate in case of contamination. Label the plate, then place into the correct incubator fir best possible growth.

Results and Discussion

RNA isolation results from nanodrop


AC

76.2

1.83

.54

AT

64.8

1.81

.40

BC

72.6

1.86

.46

BT

96.3

1.80

.53

CC

53.4

1.78

.70

CT

43.2

1.85

.61


AC

105.1

2.10

1.14

AT

64.7

2.13

1.38

BC

52.5

2.18

1.31

BT

63.8

2.13

1.59

CC

106.4

2.29

1.38

CT

21.6

2.17

1.26

After the results of the first set, it was decided to try switching to a new kit as it was suspected that the old one was contaminated, shown by the numbers in the final column, the 260/230. There is a significant decrease in contaminants, though there does still seem to be a contamination issue. For this reason, it was decided for next week that we should go through the process of ethanol precipitation to (hopefully) purify the samples.

Comments

Post a Comment

Popular posts from this blog

UV Exposure

  Intro Much of this week was dedicated to filling out our poster. What wasn't spent on poster work was spent on testing UV radiation tolerance again. The MRVP tests were done again and were again decided to be inconclusive. Method Method for UV exposure is exactly the same as last week, but R2A was exposed at levels of 1000, 1250, 1500, 2000, & 3000. TGY was exposed to 3000, 5000, 6000, 7000, 8000, & 9000. Results & Discussion Again, there was absolutely no growth on any of the R2A plates. The threshold is cleary far lower than we expected due to the media of R2A while, again on TGY, there was growth from the 3000 & 5000 plates. Conclusion Really all we have left moving forward is little details and pictures for the poster. All of the tests have been conducted at least once, but a few of them need to be redone still to see if we can verify a true positive/negative result. 
Read more

Week of 10/14/24-10/18/24

Image
Intro This week involved another round of RNA isolation, followed by purification and then an RNA gel and fluorometer check to ensure quality of the RNA before proceeding.  Methods Before RNA isolation, cells were rehydrated with 650uL of rehydration buffer per 100uL spot, then allowed to sit on the rocker for 15minutes at 250rpm to fully rehydrate. Kapton squares were then scraped to ensure removal of the cells, then pipetted into 1.5mL eppendorf tubes. RNA isolation procedure: 1. Resuspend fresh or frozen pellet in 800μl RNA lysis buffer and transfer the mixture to a ZR bashingbead lysis tube 2. Secure the tube on a bead beater fitted with a 2ml tube hold assembly and process. 1 min on/2 min ice repeated 5 times 3. centrifuge the tube for one minute to pellet debris 4. transfer up to 400μl of the cleared supernatant into a Zymo-Spin IICG column in a collection tube and centrifuge for one minute. Save the flow through 5. Add an equal volume of ethanol (95-100%) and mix well 6. tra...
Read more

Ref Gene Progress

Image
Intro     As we return to lab for the fall, we pick up exactly where we left off from the summer. With the reference gene project we've switched species to Deinococcus deserti, and with that Alex has taken time in the week inbetween to become familiarized with it. As we are officially working on the new desiccation project as well, we have been given a second species to continue our work with: Deinococcus pimensis. Over the course of the week we plated samples of both species for 24 hour, 48 hour, and 7 day desiccation.  Methods On Monday 8/19/24, a recently grown sample of deserti was gram stained to ensure no contamination before being used for inoculation. We found gram negative rods with no contaminants, so the plate was used for inoculation. A different newer plate that had left to grow over the weekend was used for starting 48hrs of desiccation to extract RNA. The 24hr and 7 day deserti, as well as the 24hr, 48hr, and 7 day pimensis were all plated the followin...
Read more