First round in.

I completed the DNA extraction protocol. On the positive side, I was able to extract a fairly large amount of DNA! On the negative side, the DNA appeared to be heavily sheared. There is a possibility that this still could generate good results with PCR, but I would rather see what I can do to reduce potential shearing points in my use of the protocol. I will also be re-evaluating the stop points I made in the protocol (for class and such), to see if I can smooth out the rather lengthy protocol and possibly eliminate points that are detrimental to the quality of the extraction.

In the above gels, you can see the faintest hint of banding. The smear is a result of DNA shearing. The lanes all contain 1μL of loading dye, and (from left to right) 2μL, 4μL, 6μL, and 8μL of the DNA extraction.

Next week I will conduct two different extractions for comparison to this protocol. One is similar to techniques I tried last semester. The other is one that I never heard of before. Matt Haberkorn came across it and told me about it. I need to do some research to find a good protocol for this extraction; it is evidently a sucrose based extraction. I will have more info on this in next weeks blog update.

Fingers crossed

This week I began working within my extraction protocol (finally). The protocol I am using is rather lengthy and this can be an issue for me as I only have certain time blocks for lab work. When scheduling my classes for this semester I tried very hard to work a non-class day into my schedule. This would have been amazing for research purposes - I would have been able to conduct this protocol all at once. Instead I have to compartmentalize the protocol. In order to be able to leave the lab lab for this semester's coursework, I have to find break points in the operation where the sample is (hopefully) stable enough to be stored until I return.

I have not fully completed the extraction as yet. I have precipitated the DNA out of solution and things look very promising at this point. There are still a few more steps for washing the sample and then storing it in a TE buffer.

Once this process is complete, I will likely run the sample through a spectrophotometer to obtain analytical data concerning concentration of DNA (thanks to Paul C. for his work on optimizing it's usage). Next will be the final tests of gel electrophoresis, and PCR.

This is a 2mL eppendorf tube containing my DNA sample. The darker yellow substance in the bottom of the tube is the DNA. My fingers are crossed in hopes that this sample will be nice and clean for purposes of electrophoresis and PCR amplification.

...

This week has found me still in a holding pattern due to the lack of the appropriate pH phenol. The good news is, it has arrived!

Through the week, I did a little research on Oak Ridge tubes for centrifugation of my samples. The tubes must be rated to withstand the action of the phenol chloroform solution. Not everything is up to the task of this. In the lab I found Oak Ridge tubes that were labeled PPCO and needed to find out if they would be appropriate. As it turns out, they are. PPCO refers to one of the constituent compounds of the tube: Polypropylene Copolymer. This is the stuff that is resilient enough to handle the phenol.

This is a screen shot from a pdf of Nalgene's catalogue 

available at http://www.catalogus.de/pdf/30/centrifugecatintl.pdf

Also, I noted that the SDS component solution for my lysis buffer had settled out of solution and essentially crystalized. This was rather worrisome for me. I know that I had followed the mixing and storage instructions to the letter. I was worried that this would in fact be a solution that I would have to prepare and use in the same day. Coupling this with a rather long extraction protocol could be very problematic! I used a magnetic stirrer on a hotplate to mix the solution for approximately 1/2 hour to no result. I was very hesitant to heat it while mixing as I didn't want to take the chance of denaturing any of the active ingredients within. Well, until I realized I will be heating it to 50 degrees C in my protocol! I applied very minimal heat while mixing and within 15 to 20 minutes there were no traces of precipitants left! 

Another hitch

Yet another hitch in my protocol.

When locating the required chemicals for my protocol, I took it on word that we had phenol on hand. Knowing someone else had conducted aDNA extraction with it, I assumed (there is the problem) that it was what I needed. What I was not aware of was the phenol available in the lab is a pH 6.6, this would be suitable for an RNA extraction. DNA extractions require a pH in the range of 7.5-8.0.

I caught this last friday, and Matt immediately ordered the appropriate phenol solution for me. It is supposed to arrive sometime in the next couple of days.

the small print is there for a reason

A fair amount of waiting time could have been avoided if I would have had the presence of mind to physically check the chemicals on hand. Lesson learned for future use.

My last few posts to this blog might convey a general tone of discouragement or dissatisfaction with my work. I thought it might be prudent of me to state this is not the case. Yes, the problems I have run into have been mildly irritating, but this is part of the process. I am still just as intrigued and interested in this as when I started out! I accept the challenges and will overcome them and hope to see some positive results by semester end.

Stay on target...

Last week, I relayed my issue concerning the concentration of proteinase k in the SDS/sarkosyl lysis buffer. Upon further research I have found several protocols that use the higher concentration (20mg/mL) of proteinase in their lysis buffer. I will be using this concentration in my first trials.

I also spent a fair amount of time researching safety protocol for sarkosyl. In higher concentrations, sarkosyl is a particularly nasty substance. The stock solution that was purchased for me is a fairly strong solution: 30%. As noted in the previous post, the "recipe" for the buffer calls for 25g of sarkosyl and as I mentioned my stock is a 30% solution. This was done intentionally, the powdered form of sarkosyl is deadly upon inhalation. Using sarkosyl in solution greatly reduces the risks associated with its handling. It does make the calculations of how much to add to get the appropriate concentration in the final solution a little more complicated, but what's a little extra math for safety's sake. While researching handling safety of chemicals is generally a good lab practice, we are being especially cautious with sarkosyl as it is a cell lysis buffer - it breaks down cell membranes, and I would like to keep mine intact.

Protocol for handling will include the usual necessary PPE: gloves (I did research solubility factors to select appropriate gloves), safety goggles, and lab coat to prevent accidental contact. I will also be handling the stock solution in the fume hood to avoid any vapor contact. The buffer I am creating will be much easier to handle as the sarkosyl will be diluted down to 5%.

I have also scaled back the overall volumes of the solutions made. For instance, the "recipe"for the 5% sarkosyl component of the lysis buffer will make 500mL. I don't need to have that much of the buffer, especially as it is untried as of yet. I have scaled back the amounts by a factor of 5, resulting in a total of 100mL. The same is being done for all of the reagents I will be using.

Looks like I am finally about to pick up where I left off at the end of last semester.