I ran a 0.8% gel prestained with Sybr Safe and viewed with the invitrogen illuminator for this stain.
And below is the image of the gel taken with my crappy-ass camera phone (Droid Bionic).
Yay! The reaction finally worked! Well mostly. It worked in lanes 6-10, which correspond to one of the reactions from the OpenPCR and 4 of the reactions from the ThermoCycler. I will assume the 5th reaction worked as well. That reaction is not visualized because there are only 10 lanes in the gel and there are 10 reactions plus the DNA ladder so one would need to be left out.
I did a reaction cleanup with Novagen PCR Cleanup. I couldn’t find my Qiaquick PCR cleanup kit and this was all I thought I had. After cleanup, I found my Qiaquick kit and will use that next time.
The nanodrop says there are 35.3ng/ul of PCR product which correlates to ~13nM. That isn’t great but it’s a start. Moving on… FINALLY!!!
This experiment is a follow up to yesterday’s experimental results. Instead of trying to program the ThermoCycler by guessing, I decided to record the temperatures of various different T settings. Basically I would pick a T and try to get the recorded value near the T’s needed for the pALS PCR protocol.
I put the machine in manual mode so I can change the T when I needed to and I would take data points at 30 seconds for melting and annealing temperatures and at 30s and 5 minutes for extending T. This is how long I would be doing that step during the PCR reaction so it seemed to make sense. Below is a chart of the Set T and the recorded T at each interval.
SO I’ve done the temp experiments and decided to do a quick temp reading of the Thermo Sprint PCR machine this morning. That data will be up shortly. The temperatures for the PCR reactions below are based on those readings, while the T for the OpenPCR reaction (also below) are based on the readings from the experiments the past few days.
Anyways now I have two machines that are better prepared to perform this PCR reaction. Both machines are set for actual temps of ~92C, 50C, 72C and hopefully I get something that I can work with today. Reaction setup is here:
Well, well, well. It looks like OpenPCR works much better than my ThermoCycler (which I just learned today is actually called PCR Sprint). The temperatures match pretty well between what the program is set for and what the recorded values are. I’ve discovered in the past that OpenPCR has a problem getting above 90C (see bottom of post), but I don’t think that is an issue here. If OpenPCR isn’t producing product then I may not have chosen the right annealing/extending temps or the problem may be elsewhere (unlikely).
ThermoCycler on the other hand is a piece of garbage. I’ve always hated this thing and now I have proof (again, I’ve done an experiment like this in the past, but it wasn’t as bad as these results show). Looking at the file “thermo-oil-3-cycles.png” you can see that ThermoCycler never gets below ~62C which is sad because it is set to anneal at 52C. It also extends at ~74C when it should be near 69C.
Tomorrow I’ll be taking some T measurements to try and get the program to be closer to the temps I want to run the pALS protocol at. OpenPCR will just need some slight modifications, but ThermoCycler needs a lot of help. Sigh…
I can only run two PCR reactions in a day, and originally I planned for four, but now I’m going to be doing 5. Why?
Well first I ran the OpenPCR pALS program with mineral oil in the tube and found out the connections were faulty so the data collection was erroneous (I would get values like -78C).
Then I ran the ThermoCycler pBSTXI program (pBSTXI is the original name for my pALS plasmid) with water in the reaction tube and the water evaporated!
Next I ran the OpenPCR pALS program again, this time with water in the reaction tube, and that water evaporated as well.
Now I’m running the ThermoCycler pBSTXI program with oil in the reaction tube to compare the results.
Tomorrow manana (that’s morning for the non-Spanish speaking) I’ll be running the OpenPCR pALS program one last time with oil in the tube again to get better data than the first run.
I’ll be publishing all the data sets at once even the bad data, because it’s open science and I can’t be open if I’m not 100% open. It’s all or nothing. Or at least that’s what my brain says the rules are.
Here is a sneak peak at the data (full data will be posted to figshare tomorrow):
Today is going to be dedicated to getting temperature readings from the PCR machines (both OpenPCR and the ThermoCycler). Both machines have their own temperature output, but in the past I’ve discovered that they don’t really reflect the temp inside the PCR reaction tube (especially with regards to the ThermoCycler). The last time I did this with the ThermoCycler, I got some interesting results which led to my current program settings.
Today I’m going to repeat that experiment and replicate it with OpenPCR. Here is my setup:
a temperature reader – I’ve used digital multimeters in the past, but for this experiment I’m using the TC-48-20 OEM because it comes with software that takes frequent measurements and allows me to export the data.
Take a PCR reaction tube and drill a small hole in it
Put either water or mineral oil in the tube (in the amount that you normally use for PCR reactions), I’m doing 50ul for this experiment.
Put the thermocouple in the tube through the drilled hole and place the tube in the block on the PCR machine.
Connect the thermocouple to your temperature reader (in my case I need to connect the temp probe to the TC-48-20 and then connect that to my computer)
I took these notes a long time ago about PCR and how to optimize for it and what each component needs. The notes are taken from the book Molecular Cloning, which is just full of amazing detail and is a must for any lab. Thought I would document this for myself (or anyone else) for that matter.
I realized that last week was a pretty wasted week. I hadn’t done PCR in a year and even though equipment shouldn’t just cease function, I should have started checking the equipment to make sure everything was working properly. I also have been just picking possible errors and adjusting instead of doing a systematic approach to correcting my PCR issues. Well my blind aiming is over and now I’m going to troubleshoot the PCR reactions the right way.