I took the following gel image on my phone and used photoshop express to enhance the contrast. All gel reactions worked. Note the smudge is a reflection of light off my filter on the illuminator.
I’m going to try to make more unzipping DNA in one final push for my PhD. Here goes nothing, I’m all in now!!!
It’s time to get back in the habit of doing molecular biology. I need to refamiliarize myself with the working PCR protocols I have, and determine if OpenPCR is more than adequate for Shotgun DNA Mapping. Using an inexpensive and open sourced platform to produce high quality research rocks my boat and I hope it does yours.
Here’s my reaction setup:
Results to come tomorrow…
Update: When adding the primers to the mastermix, I forgot which primer I added first, so I added the appropriate amounts of both. Essentially this means that there is extra amounts of one of the primers. Hopefully this doesn’t ruin the reaction, but I’m not expecting much since I’m using very old stuff here.
I’ve been writing about the OpenPCR platform for a couple weeks now and together we’ve had our ups and downs. The most prevalent issue that I’ve had in my tests is a failure to hold. By that I mean, after completing a program, a PCR reaction is usually set to hold at a certain temperature in case the user isn’t around to claim his prize (a complete reaction). I usually set the hold at 4C but discovered OpenPCR can’t handle below ~12C, but I’ve also discovered that in my case (maybe other cases) the machine doesn’t hold at all. It just ends the program and returns to room temp.
So today I investigated…
The short results: I could not consistently get the machine to hold after trying multiple programs. Nor could I get the machine to run the designated program consistently. When the machine did perform a hold in two cases there was a glitch in the LCD, but otherwise reports are ok.
Now for the long answer:
I ran 5 experiments and then gave up. I don’t like admitting that, but after a couple hours of frustrations I think it is warranted noting. Here are all my notes:
- I ran the “Simple Experiment” that is pre-programmed into the software. On my first attempt (this is after completing the PCR experiment I posted earlier), the experiment crashed. The software displayed “Done!”, while the machine sat idly. (Get used to that because this will happen a lot in this discussion.) I tried to restart and this time the machine did nothing while the software displayed the time left screen. I turned the machine off and on and tried again. This time the program ran and the machine cooled to hold after completion. While in the hold phase there was a glitch on the LCD that said “Final H” and next to the H was a rectangle and three stacked horizontal lines. (I should have taken a picture of that screen )
- I reran the “Simple Experiment.” On my first attempt the machine did nothing while the software displayed the time left screen. I turned off and on the machine. Tried a rerun and this time, the machine held at 20C. It again displayed that weird screen on the LCD.
- I created a new program: 1 cycle – 12C (for 30s), 20C (30s), 30C (30s), hold at 12C. On my first attempt, the lid heated and the software said done. Then the program started and the software started the countdown (after saying Done!). The machine got to about 15C and stayed that way for a while (time unknown) and so I stopped it. I tried to run the program again, but changed the lid heating to 0C hoping it would just not activate the lid. This time the program never started and the machine did nothing. I then changed the lid temp to 75C because I noticed it was cooling and thought maybe it needs to reach some temp before the program could start. The machine never started running.
- Another new program: 1 cycle – 37C for 30s, hold at 20C. On the first try (yay!) the machine ran, completed, and held. The LCD actually read “Final Hold” too. On a second attempt, the software said “Done!” but the experiment hadn’t started yet. It then ran and completed.
- In my last experiment, I did 1 cycle – 16C for 30s, hold at 16C. I tried twice, and both times the machine never started the program.
By that point I had given up and decided to do this write up. If I had to guess I would say there is a glitch whenever the software asks the machine to hold at anything less than 20C. There is definitely a glitch if you want to do two experiments back to back, because a lot of those times is when the machine would not do anything after completing the first of the back-to-back experiments. Other than those observations I have to give a big shoulder shrug and hope that the OpenPCR guys can give me some troubleshooting ideas. Anyone else with the system having any problems?
Today I ran the same reaction that I ran on Friday, this time I did two sets of reactions. One set, I put in the OpenPCR machine and the other set I placed in our Thermo thermal cycler. The image on the left shows the results. Lanes 1-5 contain the samples from the OpenPCR machine (Lane 5 also contains a 1kb ladder), and lanes 6-10 contain the samples from the Thermo machine. The bands are brighter towards the top than towards the bottom because I stained prior to running the gel (and the stain runs in the opposite direction from the DNA). I tried to post-stain for about 15 min to improve image quality.
Notice that the bands are all the same length and the same brightness, which was not the case in the reaction from last week. The spreadsheet of the reaction can be found below.
Here are the results of the reaction that I posted earlier today. In lane 1 is a 1kb DNA ladder. The second visible band from the bottom is 1kb in length. The next band is 1.5kb. Lanes 2-6 are each 10ul (mixed with 2ul of 6x loading buffer) from their respective PCR reaction tubes (all the same reaction). Lane 2 clearly worked the best, while lane 3 apparently contained a failed reaction. The other lanes (4-6) worked well enough in this test run. And as you can see, since the band travel distance is right between the 1kb and 1.5kb mark, that puts these fragments at right around 1.1kb (primers annealed at ~980 and 2008 on the template strand). More tests will need to be conducted in the future, but the preliminary results appear quite promising.
Note: It should be noted that this gel does not display a PCR reaction after cleanup. I went straight from the thermal cycler to the gel. The faint bands at the bottom of the image are the primers used in the PCR reaction.
This reaction is based on the reaction for making the 1.1kb anchor used in the unzipping construct, which I haven’t discussed in this space, will in the future, and some background can be found on OpenWetWare.
This particular reaction makes a 1.1kb dsDNA product that is labeled on one end with a digoxygenin molecule and has no label on the other end. The reaction can be found here.