Cheung Lab, Day 9


UMass College of Natural Sciences Research Greenhouse

Today started out by visiting the growth room on the 12th floor and selecting plants to collect seeds from.  We were collecting the T0 seeds from both arabidopsis wildtype and the feronia line.  The seed collection process uses newspaper and shaking the seed pods to release the seeds onto the newspaper.  Then you use a sifter, which allows the seeds through but keeps most of the debris from passing through.  In this way, you are able to get as many seeds as possible while minimizing the debris.


Collected seeds in eppendorf tubes
One of the most important aspects of seed collection is the potential for contamination.  You must clean up your work bench, because if seeds from a previous line enter your tube for the new line, it can be catastrophic for results, and can cause a lot of confusion in regards to results.
After we collected seeds, Norice came into lab, and we started running a gel.  We used 15 microliters instead of 10, because we were having some slight issues seeing the results earlier in the week.  We left the gel running, and analyzed other gels.  We were able to tell which of the genes were homozygous and which of the genes were heterozygous.  I haven’t read a gel in years, so this was a great review for me.IMG_20130717_130343

Gel print-outs; can you tell which plants are homozygous vs heterozygous?

We then spent time in the greenhouse in the afternoon, collecting pollen from the tobacco plants.  The process was very straightfoward- remove the tobacco flowers with the most pollen on their anthers and lay them in a tray.  I worked collecting the wildtype pollen (wildtype means “normal”) and Norice collected the pollen from the transgenic plants.  We went back to the lab with these flowers, and started to empty the pollen into eppendorf tubes.


Tobacco in research greenhouse


Collected tobacco, ready for pollen extraction in the lab.

We then tried to analyze our gel, but there were no bands on the gel.  Something had gone wrong in the PCR process, and we were not sure what it was.  It ended up being time for me to leave, so I’m not sure what could have gone wrong in our PCR.


Good example of a selection plate- the green plants are the transformed ones that survived the screening process due to antibiotic resistance.  The medium on these plates has antibiotics, which kills the other plants, which are more yellow in this picture.

Cheung Lab, Day 8


Analyzing Gel results

Today started off with a lab meeting, so I was able to head in late after cleaning my house and starting to get ready for my trip to Seattle.  I started in the laboratory by reviewing the procedures for running gels (gel electrophoresis).  The lab meeting ran late, so I was able to review the other procedures and check on the GUS assay that we set up yesterday.  I can see that the rk10p:GUS now has blue roots as well, meaning that the gene is being expressed there.

When the lab meeting ended, I started work on running gels based on the work that we did yesterday.  The first step was preparing the gels.  We used 1x TBA to set up our gels, heating the medium in the microwave and then pouring it into the gel containers to cool.  We used plastic inserts to make sure that our gels had the appropriate wells.7164

Preparing gels for gel electrophoresis

While waiting for the gels to cool, we used dye to stain the DNA in each of the containers that had come from the PCR process.  This is essential, because you want the DNA to show up so that you can view it in your gel.

We then used a micropippette to drop a very small amount of DNA into each of the wells in the gel.  This will allow us to see which of the DNA samples is larger (moves slower through gel, will be nearest to well) and smaller (moves quicker through gel, will be further from well).  DNA is negatively charged, so you place the negative end closest to the DNA wells, and the positive end away from the wells.  It is crucial to keep all of the containers organized- so that you know which well corresponds with each sample.7161

Running a gel- notice wells (blue), negative lead (black) and positive lead (red)

While running the gels, we started preparing new samples for PCR.  We first made new labels and labeled all of the plants so that we would know which sample came from each plant leaf.  We then took these leaves off of the plants using forceps and placed them into different eppendorf tubes.


Labeled plants, ready for leaf extraction

We followed the same procedure for preparing our samples for PCR that we used yesterday- the whole process takes around 3.5 hours from start to useable DNA.  In between preparing our new samples for PCR, we analyzed our earlier gels.

Cheung Lab, Day 7


View from 11th floor, Lederle Graduate Research Center

Day 7 of working in Dr. Alice Cheung’s laboratory started with us setting up a GUS assay.  GUS is a gene that we put in to visualize where something is being expressed.  In our case, we were using the plates we set up on 7/2, put in the 4 degree Celsius refrigerator for 2 days, and left in a 22 degree growth chamber for a little over a week. 


GUS assay, different genes (labeled)

To set up our GUS assay, we had to make a solution of x-glucose.  This will bind where GUS is being expressed, causing a blue color to happen.  The blue will continue to accumulate in the areas where the GUS gene is expressed until the reaction is stopped with ethanol. We started by making the solution, adding it to a Petri container, and vacuuming the air out.  Vacuuming the air out allowed more penetration of the solution containing the x-glucose, because air tends to accumulate on the edges of the plant.7154

Vacuuming out GUS assay for better infiltration

We then started learning the process for PCR- polymerase chain reaction.  PCR is used to amplify a gene that you are trying to study.  In our case, it has to do with the mutant lines of plants we are studying. First, you use a grinder to mash up the leaf and break open some of the cells.  Then, we added some extraction buffer to help extract some of the DNA.  We spun our tubes for 7 minutes, and then transferred the supernatant to a new tube.  We had to take care and not add any of the solids still left, as that would interfere with the results of the PCR.  We then added isopropanol, spun down the solution for 5 minutes, and were left with a supernatant to get rid of.  Our DNA was stuck to the bottom of our solution, so we washed whatever else was in there away with 70% ethanol.


Eppendorf Tubes getting ready for PCR

We had to resuspend our DNA in EDTA, which is a chemical that helps our reaction.  Using the vortex, we resuspended the DNA, and put it in a new microfuge tube.  Finally, after We followed the directions and set up our PCR.  We had to wait 2.5 hours for the reaction to occur.  The PCR machine was full, so we wouldn’t be able to run the gels today.

Next, Norice and I started to think about the upcoming academic year, and how we could incorporate this into our classrooms.  I started to lay out a plan, and create a plants unit that teaches most of this plant anatomy.  It’s a real shame that both the Massachusetts Science and Technology Standards (2006, latest revision), and the Next Generation Science Standards (2013), do not focus enough on plants- the basis for all food on Earth. 

Finally, we were able to check our GUS assay, and see how it would be illuminated.  The roots in our of our samples RK8p:GUS were blue, which was very interesting to see.  We will have to check the rest of the samples tomorrow and see how they turn out.

Cheung Lab, Days 5 and 6


Leaf discs after being transferred to new medium

This week was a hectic one with my Nanotechnology Class, but I was still able to work in the lab a couple of days, Monday and Friday.  It was similar to other processes that we completed earlier, so it was relatively easy on the “learning” side.

On Monday, we dipped more Arabidopsis plants with 3 different bacterial lines.  When I got to the lab, the plants were already cleaned, so it was as easy as spinning down the bacteria, preparing the solutions, and actually dipping the buds.  We then tied up the plants we had dipped the previous week.


Tied Arabidopsis plants

On Friday, we worked on transferring the tobacco leaf discs from their medium to a new medium; one that induced shoots to grow and selected for antibacterial resistance.  The process was simple- consolidating 3 plates from the previous medium to 2 plates of the new medium.  After transferring the plates, we looked at our direct-soil germinated plants, and spread them out evenly in each plant pot.



Sorting soil-germinated plants

Cheung Lab, Day 4


Arabidopsis prepped for dipping with Agrobacterium

On Day 4, our final day of the first week, we finished up learning about transformation methods.  We had a four day week because yesterday was July 4th, and the lab was closed to celebrate the holiday!

We started out the day by transferring our leaf-discs from co-cultivation medium with Agrobacterium to SIM-which is used to grow shoots.  The Agrobacterium were washed off of the leaf discs, using B5- which is basic growth medium.  We completed 3 washes to basically dilute the bacterium to a very low level.  The discs will stay in this medium for about a week.


Plates in the 22 degree Celsius growth chamber

We moved our plates to the growth chamber in the basement of Lederle Graduate Research Tower.  The plates will grow in the chamber for a while.

We then started a new process of transformation, this time using Arabidopsis.  The method we used was transformation by dipping.  Basically, you take the unopened buds, and dip them in a solution of agrobacterium.  This solution will change some of the seeds.  You then screen the next generation, find the modified seeds, and grow them into plants, which produce a new generation of modified seeds.


Mixing Agrobacterium on an orbit shaker.

Cheung Lab, Day 3


Preparing tobacco plants for a leaf transformation

On Day 3 of working in Dr. Cheung’s lab, we worked on starting a leaf disc transformation.  We used leaves from tobacco plants and cut out small discs using a leaf borer, tweezers, and scalpels.  These discs are going to be growing for about 5 weeks, using a variety of hormones.

There are multiple methods of transformation, including using Agrobacterium directly on the tissue-the method we used today, which is a live tissue-culture based method.  Transformation is the process of changing the genetic code of an organism, and creating a genetically modified organism.

It took us a couple of hours to prepare the discs, to which we added agrobacterium to transform the discs.  The leaf-discs were placed in a growth medium, and this medium will be changed on Friday.


Leaf-discs completed with Agrobacterium added

Alice Cheung’s Lab, Day 2


Our seeds, which will be ready in about five weeks.

Today, we started out the day by editing the procedure, which was different than the actual procedure that I described yesterday (and that lab members have taught us.)  We started by doing a seed screening, this time with many different genes, which we will compare in five weeks on the young plants.  The genes we were investigating were RK10p:Gus, RK11p:Gus, Wildtype (Normal) Arac7p:Gus, And Arac10p:Gus

We then prepared soil and spread seeds for direct soil germination.  There is a lab protocol for exactly how the soil is supposed to made, which was very interesting to learn (and remember well from my childhood.


A solution of nutrients to be mixed with the soil.


Our seeds are covered so that the soil doesn’t get too dry, but have a corner propped to help mitigate mold growth.

Alice Cheung’s Lab, Day 1


A lab bench with containers and seeds, Ethanol and Bleach Wash in Background.

Today, I started my first day of working for my first day in Dr. Alice Cheung’s Pollen Laboratory at the University of Massachusetts Amherst.  We started the day meeting with Dr. Cheung to discuss the schedule for the summer.  The schedule is very dynamic because I am attending (or already have attended) STEM Digital Institute at UMass Amherst, STEM Nanotechnology Institute at UMass Amherst, and the NWABR Bioethics Course, amongst my hours in working in Dr. Cheung’s lab.

We started the day learning how to do Arabidopsis screening with a T1 generation stock.  What this means is that the seeds that we are screening had an gene introduced by agrobacteria.  We use a medium with antibiotics in petri dishes, which helps us screen for the offspring that we’re looking for.  The antibiotics affect (thus killing) the normal (called wildtype) seeds, while allowing the gene that we inserted (in this case RK11p:Gus) to survive, because we added antibiotic resistance in addition to the RK11p:Gus gene.  We wash the seeds before we put them on the medium in ethanol (twice) and a 50% bleach solution with detergent (once).  We then use a laminar flow bench to maintain a sterile environment, while rinsing the seeds with double-distilled water (4 times).


We use this contraption to shake the seeds during the rinses.

To standardize germination, the seeds are stored at 4 degrees celsius for 2 days- making the seeds believe it is winter.  Then, when they are transferred to the growth chamber (at 22 degrees Celsius), a more uniform sprouting occurs.

We prepared a number of RK11p:Gus plates, which we will be transferring to the growth chamber on Wednesday.


Laminar Flow Bench for a sterile environment.


Eric Johnson in front of a growth chamber in the basement of Lederle Graduate Research Tower.