Finding Parts from Jakku

This week I completed another extraction with the ZR Plant/Seed DNA MiniPrep™ kit, but included an overnight incubation in room temperature and an extra 10 minutes in a man-mad bead beater. Josh was able to come up with a new way of bashing my beads by creating an awesome "home-made" bead beater here in the lab. Choosing between buying an actual bead beater or creating one, Josh's superior sagacity thought up of creating one. I was able to bash my beads without the numbness taking over my hands and the beating of the beads would be consistent. I saw improvement in the bashing of the cholla spines and the solution actually turned a bit yellow. This did not happen previously with my first extraction and I am hoping it was a serious improvement. I'll see if there are any bands next week after the samples undergo electrophoresis.

The Awakening Begins

Hello all! Last semester I was trying out a new project, other than amplifying ant DNA my last spring semester and summer. I decided to try to amplify DNA from a new source such as plants! The protocols for extracting DNA from ants and plants are completely different from one another. It was a difficult process trying to extract DNA from ants, and I was glad to have heard about a new kit I will be using. The kit is called ZR Plant/Seed DNA MiniPrep™, and their website claims the process will take only fifteen minutes. I am hoping this is true because I will be trying to extract from the spines of cholla cacti. I will specifically try to extract DNA from teddy-bear cholla. If you want to find out more about what kit I will be using in the lab feel free to check out the link below!

 https://www.zymoresearch.com/dna/microbial-environmental-dna-isolation-1/soil-fecal-plant-dna/zr-plant-seed-dna-miniprep

I will be conducting this experiment carefully because I do not want to end up looking like this.

Thanks to the Deathstar

This week was pretty hectic working on our SSTEM projects as well as our final draft. I tried running some more gels this week in hopes of seeing some DNA from my newest extractions. My gel still did not show any DNA from under the UV lighting. In my presentation I felt a bit afraid of showing gels without polymorphic banding, but my mentors/SSTEM scholars reassured me that it was not a waste. Through my failure of not extracting DNA from the cholla spines actually proved those protocols I used were not effective and to avoid using the same steps next time.

 One of my gels from this past week that showed I had not yielded any DNA from my extractions. In my next post I hope to have some polymorphic banding.

Going Forward On A Speeder Bike

Fortunately, I was able to conduct these DNA extractions in the biology lab and I was able to retrieve a piece of cylindropuntia bigelovii from the PC greenhouse. The next steps will be to create different reagents in order to extract the DNA from the cholla spines. I also plan on collaborating with other DNA plant extractions I have read from previouse S-STEM scholars in hopes of having some luck on extracting DNA and seeing it under the UV light after electrophoresis. I need to see polymorphic banding from cholla cacti in order to answer my question on whether chollas reproduce sexually or asexually.

Stuck in the Deserts of Tatooine

During the week of November 19th, I was still working on my extractions and began to run gels on them. I figured that if I had my samples incubate overnight in the lysis buffer the fatty membranes and proteins associated with the DNA would have broken down. I also thought I would have seen a DNA pellet form in the -20 degree isopropyl-alcohol after waiting 30 minutes, but I was not able to see anything form. After extracting most of the isopropyl-alcohol from the microcentrifuge-tube, I allowed it to air-dry underneath the hood. I then suspended the DNA in distilled water. My samples underwent electrophoresis and here is the gel:

 This gel held three samples from my three separate DNA extractions. I was not able to see any DNA under the UV light. It was not something I had hoped to see.

Beginning Life on Alderaan

During the week of November 12, I continued trying to perfect my Cacti DNA extraction. This new protocol involved my older protocol to extract DNA from ants. This protocol involved:

• I put 5-15 spines into a microtube and wash with bleach.
• I then decant and rinse the spines twice with distilled water.
• I cut the spines into smaller pieces with nail-cutter scissors.
• I add 90 micro-liters of nuclease free water, 1 micro-liter of 10% SDS, 2 micro-liters of 20% meat tenderizer solution.
• I heated the tube at 75 degrees Celsius for 10 minutes in a water bath and agitated the tube thereafter with a flick of my finger.
• I heated the tube at 95 degree Celsius for 5 minutes in a heating block.
• I agitate the tube with a flick of my finger then allow the tube to incubate in room temperature overnight.
• I take the sample and put it into the centrifuge for 13.2 rpm for 5 min.
• I take the sample out of the centrifuge and remove the supernatant from the top in order to transfer into a new microcentrifuge-tube. 
• I add -20 degrees Celsius isopropyl-alcohol equal to the amount of supernatant and put the tube at -20 degrees Celsius for 30 minutes.
• I centrifuge the sample and collect the supernatant into a new tube.

The heating block used and the other picture shows two samples with -20 degree Celsius isopropyl-alcohol.

Beginning A Trek Into A New Naboo

During the week of November 1st, I began the protocol for extracting DNA from the cholla cacti. I received a piece of cholla from our green house in hopes of extracting DNA and finding clear polymorphic banding after running the DNA sample in a gel. There were various ways on how I began isolating the DNA.

• The first protocol I used consisted of washing 5-15 spines in a microtube and washing them in bleach and decant with a disposable pipet.
• I rinse the spines with distilled water twice and decant.
• I cut the spines with nail-cutter scissors.
• I added 1000 micro-liters of lysis buffer after the spines are cut and incubate for 30 minutes at 65 degrees Celsius. 
• I disrupt the spines by vortex machine and incubate for another 30 minutes.
• I then incubate the sample overnight by allowing it to sit at room temperature.
• I add 5 drops of protease to the microcentrifuge-tube and heat for 10 minutes at 65 degrees Celsius.
• I vortex the sample and put it in the centrifuge at 13.2 rpm for 4.0 minutes.
• I take the sample out of the centrifuge and remove the supernatant from the top in order to transfer into a new microcentrifuge-tube. 
• I add -20 degrees Celsius isopropyl-alcohol equal to the amount of supernatant and put the tube at -20 degrees Celsius for 30 minutes.
• I centrifuge the sample and collect the supernatant into a new tube.