Difference between revisions of "Team:US AFRL CarrollHS/Notebook"

Line 155: Line 155:
 
treat. All 12 days of hard work deserved a celebration and the students and mentors
 
treat. All 12 days of hard work deserved a celebration and the students and mentors
 
bonded over frozen custard and ice cream.</p>
 
bonded over frozen custard and ice cream.</p>
 +
</div>
  
 +
<div class="text2">
 +
<h2>Week 4: June 19-23</h2>
 +
<h3>Monday (06/19/17)</h3>
 +
<p>After the weekend, the students met in the lab and initiated a second
 +
digest. They were determined to get everything measured correctly and
 +
leave minimal room for mistakes. This time around, the students were
 +
much more knowledgeable about buffers and enzymes used and
 +
proceeded carefully combining them with DNA. After the digest was complete, the
 +
young scientists began prepping another gel for electrophoresis. Everything went
 +
smoothly while the digest was completed and DNA was extracted and cut by the
 +
enzymes. Next, the students prepped the DNA by adding a loading dye and then
 +
proceeded to put it on the gel. After electrophoresis for 30 min, the gel was complete.
 +
The LabPats were able to use this downtime for updating lab journals, creating new
 +
and exciting logos, as well as continuing to work on the wiki. Next, another
 +
experienced mentor helped with analyzing the gel and cutting out the DNA from the
 +
gel. However, they were quite disappointed to learn that there were 3 bars, indicating
 +
more base pairs than expected. The mentors ensured them that the gel still worked
 +
and that they just needed to cut out the fluorescent band that was relevant.
 +
Meticulous work was done to cut out the gel, and the labpats were finally able to let
 +
out a sigh of relief once the gel was safely placed in the tube and put in the fridge to
 +
be melted down tomorrow.</p>
  
 +
<h3>Tuesday (06/20/17)</h3>
 +
<p>The students quickly resumed where they had left off to begin their gel
 +
extraction. Two different protocols were used to see which one would
 +
achieve a higher concentration. The teams were divided into Tina and Annie
 +
working with Svetlana on (QIAEX II Agarose Gel Extraction) and Jason and Dallas (who
 +
unfortunately was ill, so Annie and Tina tag teamed his sample) working with Mike on
 +
QIAEX Gel Extraction Kit. The gel extractions were performed and the nano drop test
 +
was performed. The only successful gel extractions were performed by the girls
 +
(Svetlana’s protocol), so they took home the victory! The boys learned a lot from their
 +
error and submitted defeat. The day was wrapped up by working hard designing the
 +
wiki and learning how to code.</p>
 +
 +
<h3>Wednesday (06/21/17)</h3>
 +
<p>Today the students began their second Polymerase Chain Reaction (PCR).
 +
Under the watchful eye of Dr. Harbaugh, the students independently finished
 +
the PCR and loaded it into the thermocycler. Another small gel was prepared
 +
and the PCR’s were taken out and loaded on the gel. The students meticulously loaded
 +
the PCR’s onto the slits of the gel with incredible precision that truly showed their
 +
growing expertise.</p>
 +
<p>The students received the results they had been hoping for, which showed that the
 +
Super folded GFP had been inserted into the bacteria. They then performed a PCR
 +
Purification, which separated the massive amounts of DNA. The DNA was then
 +
measured using the nano drop and these results were given:</p>
 +
 +
<h3>Thursday (6/22/17)</h3>
 +
<p>The day began with another PCR. Today, the students were ready to
 +
completely do the PCR by themselves with little guidance from the mentors.
 +
The standard protocol was followed and the PCR and ran another cycle in
 +
the thermocycler. Meanwhile, another gel was prepared by the students. This time
 +
around, the students were very efficient and the process went by very quickly. The gel
 +
ended up giving too many bands, but the mentors conversed and decided to proceed
 +
with the PCR purification anyways. After the lab work was done, the students decided
 +
to meet with the other lab students to discuss current progress. The students created
 +
discussion points and had a great discussion with the other lab.</p>
 +
 +
<h3>Friday (6/23/17)</h3>
 +
<p>The day began bright and early with two PCR digests. This time a PCR
 +
Vector Digest was conducted on the same from yesterday and a PCR GFP
 +
digest was done on the sample from Wednesday (6/21/17). After following
 +
the protocol, the students were able to successfully perform the digests. After
 +
a 2 hour wait (per the protocol), the young scientists performed a purification on the
 +
GFP digest. The goal was to cut off end bits of the base pairs to allow the plasmid
 +
connect again. During the students' lunch break, another meeting was held with teacher
 +
leaders who wanted to know information about progress and future proceedings.</p>
 
</div>
 
</div>
 +
<div class="text">
 +
<h2>Week 5: June 26-30</h2>
 +
<h3>Monday (6/26/17)</h3>
 +
<p>The students began another week of hard work. They made a gel to run
 +
their previous Plasmid Digest. Next, they looked at the gel under the blue
 +
light and found the band they were looking for at 3.2k base pairs. Next they
 +
scalpeled out the desired band and began a gel extraction using the QIAEX ll
 +
Agarose Gel Extraction Protocol. After the gel extraction they tested their results on the
 +
Spectrotropsesis. The first sample was lower than preferred and the next sample had a
 +
very uneven graph, but with the desired numerals. Afterwards, the mentors discussed
 +
and determined to still do a legation the next day and see the results.</p>
  
 +
<h3>Tuesday (6/27/17)</h3>
 +
<p>Today was the student's first ligation. The goal was to put the sticky ends
 +
back together to form a complete plasmid. A lot of math was done to
 +
determine the exact ratios of sfGFP and PCR vector plasmid. Luckily, Dr.
 +
Harbaugh was able to assist the students with newly introduced lab techniques. There
 +
was another protocol and the students were able to follow it with no problem. They
 +
then went to the conference room and had another meeting with the other labs and
 +
teachers. This time, both labs gave extraordinary presentations about project progress
 +
and end goal. The RH lab presented their abundance of lab work and myriad of
 +
website/wiki work. The RX lab presented lab work and some technical website work.
 +
Afterwards, the groups planned out agenda and discussed necessary steps to
 +
complete the wiki page.</p>
 +
 +
<h3>Wednesday (6/28/17)</h3>
 +
<p>The students began the day with a new PCR. After examining the plates from
 +
yesterday, it was determined that there indeed was green fluorescence on the
 +
plates. However, a PCR was needed with a gel to determine whether or not it was
 +
sfGFP or GFP. High in spirits, the students immediately went to work. First, they
 +
created a Master Mix following the PCR Protocol for Phusion DNA Polymerase. They
 +
did some tricky math and figured out exact proportions needed for nuclease-free water,
 +
Phusion Buffer, DMSO, Kpn1(forward primer), BsrG1(reverse primer), as well as the
 +
phusion DNA polymerase. They each used 6 colonies. Four students used green
 +
colonies and then dipped it into the PCR and the LB Broth with chloramphenicol. This
 +
method ensures that only the bacteria immune to chloramphenicol can survive. On the
 +
gel, it should give a precise band of base pairs indicating sGFP or GFP. Another
 +
student used 6 colonies without the green fluorescence. It acts as a control. When it is
 +
run as a gel, there should be random bars that indicate there is no sGFP or GFP. Then
 +
the samples were put into the thermocycler for 1.5 hours according to the protocol.
 +
The students then had the endure a nerve raking wait as they wondered if the PCR had
 +
gone successfully and whether the bacteria had sGFP or not. After preparing another
 +
gel, the students meticulously put the samples on the 40 wells. After 30 minutes of
 +
running the gel, the students came back to an ecstatic Dr. Goodson informing them
 +
that the gel had turned out as planned and that they could proceed with the mini-prep
 +
before sending in the DNA for sequencing. Everyone shared "elbow-fives" and were
 +
relieved that all their hard work had not gone to waste!</p>
 +
 +
<h3>Thursday (6/29/17)</h3>
 +
<p>Today the students began to mini-prep the samples from the previous day.
 +
Right from the start, the students noticed fluorescent green coming from
 +
one of the tubes. This would foreshadow what they would see later in the
 +
day. They began the mini-prep full of hope and concentrated on following the protocol
 +
exactly to achieve the best possible results. They decided to first put the tubes over
 +
the light and observed a lot of fluorescence (see picture). After about 2 hours, they
 +
were finally done. They nano dropped the sample and observed an incredible amount
 +
of DNA. Everyone cheered as they realized that the "response" component of the
 +
project was successful.</p>
 +
 +
<h3>Friday (6/30/17)</h3>
 +
<p>Since all the lab work is currently done until the DNA sample is sent to Ohio State
 +
University for DNA sequencing, the students had the whole day to make website
 +
improvements and wiki renovations. New logos were also created as well as
 +
graphics. The students will continue to add content to the wiki weekly.</p>
 +
</div>
  
 
</html>{{US_AFRL_CarrollHS_Content_End}}{{US_AFRL_CarrollHS_Nav}}
 
</html>{{US_AFRL_CarrollHS_Content_End}}{{US_AFRL_CarrollHS_Nav}}

Revision as of 20:39, 29 October 2017


Notebook

A detailed description of what we did in the lab everyday!

Week 1: June 1-2

Thursday (06/01/17)

Exciting first day orientation and safety training off-Base for the newly assembled crew. The mentors and students bonded as work started towards a common goal.

Friday (06/02/17)

On the second day, the students began learning and overviewing basic lab techniques. These techniques include: conversion factors, serial dilutions, micro pipetting, and creating and pouring plates of LB agar. Under the tender supervision of knowledgable mentors, the students learned to accurately master lab techniques that would prove invaluable once lab work began.

Week 2: June 5-9

Monday (06/05/17) and Tuesday (06/06/17)

The first two days of this week were spent on Wright Patt Air Force Base (WPAFB). The team members toured the two labs that they would be working in and received safety training in both. The student researchers were very excited to tour the world-class facilities on base. Multiple brainstorming discussions were also held to finalize the project idea. After the week of training, the group of students split up into 2 groups: one to work in the 711th Human Performance Wing, and one to work in the Materials Directorate. The young researchers at the 711th Human Performance Wing were tasked with sensing and response while the researchers at the Materials Directorate were tasked with packaging.

Wednesday (06/07/17)

On the first day in the lab the members made LB broth, (formula in protocol tab). Although a simple procedure, this was the first hands-on experience for the students in the lab. They then did a plasma transformation protocol using E.coli JM109 and the plasmid PRhl_GFPa1. The bacteria created was plated onto LB chloramphenicol and left in incubator (37°C), overnight. If there was growth on the plates in the morning there would a greater probability that the E. coli absorbed the plasmid. The tired but excited young researchers went home with fingers crossed.

Thursday (06/08/17)

When they entered the lab on the second day the plates showed growth with distinctions of individual colonies. Mentor Mike Goodson ecstatically let the students know about the significance of the growth and students were relieved that their plates had grown! These colonies were used to complete the PCR protocol. The master mixture, (protocol tab) and LB broth in round bottom falcon tubes were left in the incubator (37°C), overnight. The plates were put in the refrigerator (4°C) to preserve the bacteria for future use. This protocol was used to confirm that E.coli JM109 had absorbed the plasmid PRhl_GFPa1

Friday (06/09/17)

For the last day of the week, the students’ goal was to confirm the E. Coli had absorbed the plasmid and then perform a MiniPrep Kit to take the plasmid out of the bacteria. To accomplish this, they first learned how to create a gel and how to use the electrophoresis. The electrophoresis sends the DNA down the gel using a positive and negative charge. Under a blacklight you can see if the DNA is present. Next, the students use a QIA prep spin Miniprep Kit to take plasmid out of bacteria. The students placed one nanodrop onto spectrophotometer to measure how pure the DNA extracted is. For future experiments, the plasmid DNA would then be sent to an offsite lab to be sequenced.


Week 3: June 12-16

Monday (06/12/17)

The students went to a group meeting within their labs. Students streaked bacteria onto agar plates and put back into the incubator. The rest of the day was spent collaborating on website design and graphic design. Two coding novices, Annie and Jason, worked on learning html, CSS, and Javascript. Graphic designer Tina created a new mascot microbe. Assistant graphic designer and artist Annie created a drawing of the original Wright Flyer and assisted in Photoshop drawing. Stenographer Hayley typed up protocols and helped organize team documents.

The whole team met in the morning to discuss the Wiki and each group’s project. The RX team observed the preparation and running of PCR. The team made LB broth and agar. Later the team observed the purification of the PCR results and the analyzation of the results with Qubit.

Tuesday (06/13/17)

-The students removed the agar plates with bacteria from the incubator. Two tubes were filled with LB broth, one labeled as a blank and one containing the bacteria. Using a sterile toothpick, a single colony was picked from each plate, and placed in the culture tube. An unused sterile toothpick was added to the blank tube, to verify that the process was sterile. These tubes were then shaken in an incubator for 3 hours.

The students conducted an experiment using culture growth to see if the bacteria will turn green with the addition of the quorum sensing molecule to the bacteria.

The next morning, the plasmid should glow green indicating it is present.

Wednesday (06/14/17)

The students retrieved the test tubes placed in the incubator the day before and analytically checked the optical density and the fluorescence of the colonies. Immediately afterwards, a visual check was performed as well, confirming that the Colony + 3OC12 glowed green. The ecstatic students celebrated as anticipated results showed through the light.

In the afternoon, the students were back at work again preparing a mini-prep on the PLCD-GFPa1 (the plate streaked on Monday). The remaining DNA containing the GFP was then placed in the -20o C freezer for storage. Having much more experience the second time doing the mini-prep, the trailblazing students had a much better grasp and spent less time with more accuracy.

Thursday (06/15/17)

-Time to go back to work bright and early. A little after the crack of dawn, the engaged students immediately went to work preparing a digest. The scholars made a 15 uL digest along with a Master Mix. Interactive steps included preparing a mini-prep, mixing with enzyme BSr61-HF, enzyme KpuI-HF, buffer Cutsmart, and water. The remaining solution was then placed in incubation for 2 hours. In addition, students were excited to read and learn about plasmid cloning by restriction enzyme digest and restriction endonuclease. With a desire to learn more, the students trekked back to the lab and met with another qualified scientist to assist in the digest. Once the digest was done, the students made a gel and electrophoresis to analyze the DNA. The electrophoresis sends the DNA down the gel using a positive and negative charge and allows the students to see the amount of base pairs, and in this case also allows the students to see the cut DNA and replacement. However, the young scientists were very disappointed to find out the the digest did not go as expected and DNA did not get replaced as expected. Disappointed but not defeated, the students immediately went back to work and retraced their steps, beginning the process all over again. Things do not always work out in the lab and the students were eager to restart and achieve favorable results.

Friday (06/16/17)

-Immediately the next day, the eager students were right back at work. The previous day's failure had fired them up and they were eager to do everything carefully and quickly. The day began with a student-led mini-prep in which the students were able to follow the protocol and do everything by themselves. Afterwards, the students and mentors went out for a cool ice cream treat. All 12 days of hard work deserved a celebration and the students and mentors bonded over frozen custard and ice cream.

Week 4: June 19-23

Monday (06/19/17)

After the weekend, the students met in the lab and initiated a second digest. They were determined to get everything measured correctly and leave minimal room for mistakes. This time around, the students were much more knowledgeable about buffers and enzymes used and proceeded carefully combining them with DNA. After the digest was complete, the young scientists began prepping another gel for electrophoresis. Everything went smoothly while the digest was completed and DNA was extracted and cut by the enzymes. Next, the students prepped the DNA by adding a loading dye and then proceeded to put it on the gel. After electrophoresis for 30 min, the gel was complete. The LabPats were able to use this downtime for updating lab journals, creating new and exciting logos, as well as continuing to work on the wiki. Next, another experienced mentor helped with analyzing the gel and cutting out the DNA from the gel. However, they were quite disappointed to learn that there were 3 bars, indicating more base pairs than expected. The mentors ensured them that the gel still worked and that they just needed to cut out the fluorescent band that was relevant. Meticulous work was done to cut out the gel, and the labpats were finally able to let out a sigh of relief once the gel was safely placed in the tube and put in the fridge to be melted down tomorrow.

Tuesday (06/20/17)

The students quickly resumed where they had left off to begin their gel extraction. Two different protocols were used to see which one would achieve a higher concentration. The teams were divided into Tina and Annie working with Svetlana on (QIAEX II Agarose Gel Extraction) and Jason and Dallas (who unfortunately was ill, so Annie and Tina tag teamed his sample) working with Mike on QIAEX Gel Extraction Kit. The gel extractions were performed and the nano drop test was performed. The only successful gel extractions were performed by the girls (Svetlana’s protocol), so they took home the victory! The boys learned a lot from their error and submitted defeat. The day was wrapped up by working hard designing the wiki and learning how to code.

Wednesday (06/21/17)

Today the students began their second Polymerase Chain Reaction (PCR). Under the watchful eye of Dr. Harbaugh, the students independently finished the PCR and loaded it into the thermocycler. Another small gel was prepared and the PCR’s were taken out and loaded on the gel. The students meticulously loaded the PCR’s onto the slits of the gel with incredible precision that truly showed their growing expertise.

The students received the results they had been hoping for, which showed that the Super folded GFP had been inserted into the bacteria. They then performed a PCR Purification, which separated the massive amounts of DNA. The DNA was then measured using the nano drop and these results were given:

Thursday (6/22/17)

The day began with another PCR. Today, the students were ready to completely do the PCR by themselves with little guidance from the mentors. The standard protocol was followed and the PCR and ran another cycle in the thermocycler. Meanwhile, another gel was prepared by the students. This time around, the students were very efficient and the process went by very quickly. The gel ended up giving too many bands, but the mentors conversed and decided to proceed with the PCR purification anyways. After the lab work was done, the students decided to meet with the other lab students to discuss current progress. The students created discussion points and had a great discussion with the other lab.

Friday (6/23/17)

The day began bright and early with two PCR digests. This time a PCR Vector Digest was conducted on the same from yesterday and a PCR GFP digest was done on the sample from Wednesday (6/21/17). After following the protocol, the students were able to successfully perform the digests. After a 2 hour wait (per the protocol), the young scientists performed a purification on the GFP digest. The goal was to cut off end bits of the base pairs to allow the plasmid connect again. During the students' lunch break, another meeting was held with teacher leaders who wanted to know information about progress and future proceedings.

Week 5: June 26-30

Monday (6/26/17)

The students began another week of hard work. They made a gel to run their previous Plasmid Digest. Next, they looked at the gel under the blue light and found the band they were looking for at 3.2k base pairs. Next they scalpeled out the desired band and began a gel extraction using the QIAEX ll Agarose Gel Extraction Protocol. After the gel extraction they tested their results on the Spectrotropsesis. The first sample was lower than preferred and the next sample had a very uneven graph, but with the desired numerals. Afterwards, the mentors discussed and determined to still do a legation the next day and see the results.

Tuesday (6/27/17)

Today was the student's first ligation. The goal was to put the sticky ends back together to form a complete plasmid. A lot of math was done to determine the exact ratios of sfGFP and PCR vector plasmid. Luckily, Dr. Harbaugh was able to assist the students with newly introduced lab techniques. There was another protocol and the students were able to follow it with no problem. They then went to the conference room and had another meeting with the other labs and teachers. This time, both labs gave extraordinary presentations about project progress and end goal. The RH lab presented their abundance of lab work and myriad of website/wiki work. The RX lab presented lab work and some technical website work. Afterwards, the groups planned out agenda and discussed necessary steps to complete the wiki page.

Wednesday (6/28/17)

The students began the day with a new PCR. After examining the plates from yesterday, it was determined that there indeed was green fluorescence on the plates. However, a PCR was needed with a gel to determine whether or not it was sfGFP or GFP. High in spirits, the students immediately went to work. First, they created a Master Mix following the PCR Protocol for Phusion DNA Polymerase. They did some tricky math and figured out exact proportions needed for nuclease-free water, Phusion Buffer, DMSO, Kpn1(forward primer), BsrG1(reverse primer), as well as the phusion DNA polymerase. They each used 6 colonies. Four students used green colonies and then dipped it into the PCR and the LB Broth with chloramphenicol. This method ensures that only the bacteria immune to chloramphenicol can survive. On the gel, it should give a precise band of base pairs indicating sGFP or GFP. Another student used 6 colonies without the green fluorescence. It acts as a control. When it is run as a gel, there should be random bars that indicate there is no sGFP or GFP. Then the samples were put into the thermocycler for 1.5 hours according to the protocol. The students then had the endure a nerve raking wait as they wondered if the PCR had gone successfully and whether the bacteria had sGFP or not. After preparing another gel, the students meticulously put the samples on the 40 wells. After 30 minutes of running the gel, the students came back to an ecstatic Dr. Goodson informing them that the gel had turned out as planned and that they could proceed with the mini-prep before sending in the DNA for sequencing. Everyone shared "elbow-fives" and were relieved that all their hard work had not gone to waste!

Thursday (6/29/17)

Today the students began to mini-prep the samples from the previous day. Right from the start, the students noticed fluorescent green coming from one of the tubes. This would foreshadow what they would see later in the day. They began the mini-prep full of hope and concentrated on following the protocol exactly to achieve the best possible results. They decided to first put the tubes over the light and observed a lot of fluorescence (see picture). After about 2 hours, they were finally done. They nano dropped the sample and observed an incredible amount of DNA. Everyone cheered as they realized that the "response" component of the project was successful.

Friday (6/30/17)

Since all the lab work is currently done until the DNA sample is sent to Ohio State University for DNA sequencing, the students had the whole day to make website improvements and wiki renovations. New logos were also created as well as graphics. The students will continue to add content to the wiki weekly.