Vena Haynes's blog

This final week was mostly spent working on my final paper and final presentation.  I also made more dsDNA targets to increase Sharp's stock.  One of my summer goals was to compare ssDNA and dsDNA target data, but there was not enough time to use the ssDNA targets I prepared.  Luckily, I will be returning in the fall as a Masters student, and will hopefully be able to continue on with this project. 

Last week I accomplished my initial goal at Sharp.  I set out to show the applicability of these impedance sensors to be used on genomic assays.  To do this, I used three E.

This week I tried to make single-stranded targets by running double-stranded PCR and then using a digestive enzyme (endonuclease) to end up with a single-stranded product.  However, the initial PCR reaction did not work, possibly due to degraded template. 
The purpose for single-stranded targets is to get a comparision between ds and ss targets.  ds targets are economically more viable because they take less time to produce.  ss targets are the standard, and so if we can show that ds data matches or is close to ss data, then we have optimized DNA assays.

All day Monday I worked on my final report and read more primary literature.  Tuesday and Wednesday I was at Sharp and continued to test different parameters on the biosensors.  Some of the sensors were functionalized (probes attached to the surface) alongside of a synthetic molecule called MCH that previous literature reported would decrease the amount of non-specific binding of the targets to the probes.  The little preliminary runs that I performed did not show this.  I did find some optimal parameters for the biosensors:  2XSSPE buffer, 75 Hz, 75 mV.

This last week was pretty basic.  I continued to try new DNA and buffer concentrations on the biosensors, and found that 0.5µg of ds DNA target works the best.  This is promising because it is the lowest concentration we tried, meaning that our samples will last longer.  The data on the different buffer concentrations was inconsistent and therefore inconclusive.  This week we will try different temperatures because the 52°C we have been using for double-stranded targets may affect the adhesive on the channels after multiple runs.  

This week I was at SHARP and helped in the optimization of the impedance sensors.  Due to inconsistent data, different measures are being taken to figure out the problem.  We first began running the sensors at different frequency settings, and are now changing the concentrations of buffer and DNA targets, as well as different temperature controls.  This optimization usually is done using short oligonucleotide strands, but some of the chips were run with ds DNA targets I created here at CMOP. 

This past week I spent two days at SLA doing quality control of the biosensors by either running oligonucleotides or double-stranded DNA targets and testing variable frequency settings.

At CMOP I made more double-stranded DNA targets and purified the PCR products.  I purified my RNA extractions, and I started the process of making single-stranded DNA targets...but have yet to purify them.

The week ended with a great trip to Bonneville Dam and a hike up Multnomah Falls which was awesome. :)

-Vena

This week I spent my week at Sharp Labs of America (SLA) and was able to see first hand the biosensors I am helping design.  It was very interesting to be able to see the gold surface of the electrodes under a scanning electron microscope.  There has been some complication with the cleaning of these biosensors, and so various treatments were performed, and my goal this week was to test the biosensors using oligonucleotides to see which treatment works best (doesn't disrupt the gold surface, but effectively cleans off a previous run).

This week I continued to do PCR of our target dsDNA.  Two of our genes had clear enough bands and were the right length to continue on with purification, and so I purified our PCR samples and determined the DNA concentration.

Because we are waiting on an order of new primers, I began RNA extraction of ocean water samples.  The first time I assisted in the extraction, and the next two times I performed the extraction on my own.  The next step with our RNA pellet is to completely purify by removing any DNA present with DNase.

This week has been a lot of reading, reading, re-working PCR, and more reading.  I feel somewhat accomplished as I can say I have a grasp on the project I am involved in, and my individual role.  I am involved in designing more efficient and cheaper real-time sensor array platforms that do not require flourescence labeling.  These sensory arrays will hopefully be applicable to environmental sampling.  In order to test the quality and efficiency of these sensors, target DNA (both double and single-stranded) samples need to be made.