Spring 11:QRT-PCR: Progress Notes

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To-Do

Last updated 23:01, 5 May 2011 (EDT)

• fine-tune Tlid involvement in the model (fix it so it doesn't affect whole graph)
• reduce lid heater current so it doesn't melt the tube

Weekly "goals"

as of 15:03, 4 April 2011 (EDT)

• 3/25: Optics finished. Code started.
• 4/1: Heated lid finished.
• 4/8: No really, heated lid finished. Updated heat transfer function. Kinda know LabView.
• 4/15: Successful PCR.
• 4/22: Optics are ready for QRT-PCR!
• 4/29: Successful QRT-PCR from the side.
• 5/6: QRT-PCR from the top. Now which one was better?

Notes

5/8/11

Posted 14:27, 17 May 2011 (EDT)

Ran a PCR. Used parameters as noted on 5/5/11

Commercial machine: program NK1:

• 95C for 30 sec
• repeat x 30:
  • 95C for 30 sec
  • 55C for 1 min
  • 72C for 10 sec
• 4C for infinity

345 machine: 30 cycles

• 94C for 3.8 min
• repeat x 30:
  • 95C for 100 sec
  • 54C for 65 sec
  • 72C for 70 sec
• 4C for infinity.

began commercial at 3:05; 345 machine at 3:17

Observations:

• Commerical: Tube I used was too tall, and so was misshapen upon removal. It also took longer than expected, for some reason, and was halted early.
• 345: Cap of tube was melted a bit. Was left at 4C for about an hour, which may have caused the condensation on the heating block. Ended earlier than expected - what happened?

Checked product on a gel:

5/7/11

Prepped to run PCR.

5/5/11

Posted 23:01, 5 May 2011 (EDT)

Added a gain term to the temperature model, that kinda factors in T_lid. Now the model is a lot better; within 1-2 degrees! Which means we're ready to do a PCR next time. yay.

Parameters that result in a good model:

• Gain=0.15
• I_lid = 1.95
• K_p = 1.2
• K_i = K_d = 0
• a_zo = 11.1
• a_z1 = -9.9
• b_z = 1

5/1/11

Posted 16:46, 1 May 2011 (EDT)

Yesterday I made sure all the thermistors were working. Today I made sure the resistance wire was working.

Temperatures are correct in v2.712. Only the Lid temperature (which I just added) is correct in v2.52, which I need to take the frequency data. Will seek further advice from Steven tomorrow.

I am also not sure what exactly we want from our lid heater.

4/27/11

Posted 17:59, 27 April 2011 (EDT)

FUN FACT: you can strip a thermistor wire using a razor.

Cleaned out the lid so it can be used. Then, I soldered the Lid Heater Thermistor and Sample Thermistor. I also fixed the resistance wire that broke. The lid is not hooked up, but it is ready. I wish I had been able to work faster and do more, but... I'll be back tomorrow.

4/26/11

Posted 17:59, 27 April 2011 (EDT)

Drilled a hole in the lid for the Lid Heater Thermistor. Poured epoxy, which is now curing. I had planned to put a washer on top of the epoxy to keep it flat, but it wouldn't fit in there (thanks to the slight tilt of the rod). So we'll hope for the best, and use RTV to flatten it if all else fails.

4/20/11

Posted 19:31, 20 April 2011 (EDT)

OK heated lid is pretty close to being actually usable. Maybe. There are holes in the sides of the cube so we can hold the resistances wires straight. This is good, because otherwise the wires melt the plastic.

The lid screws on to the base well, if you use the cut-off lens tube.

The copper wire is kept flat thanks to some RTV. It's rated for up to 150C.

Problems:

• lid does not fit inside cube thing. lid is too tall. For now this has been fixed with some black cardboardy stuff.
• I broke the sample thermistor. Whoops. :(
• the hole in the thermal epoxy is not quite centered, so you don't see as much of the sample as you could from the top. Eventually I should make a new lid to fix this....

4/6/11

Posted 17:13, 6 April 2011 (EDT)

Learned some more about LabView.....

I've constructed the heated lid. Now I just need to hook it up electrically. Which I can't do...without some guidance, since you gotta use something called acid flux with the NiChrome. okay.

The heater in the lid is a little too tall, so that the lid doesn't quite fit under the cap. Probably smaller-diameter washers could fix this. But for now, it doesn't need to be fixed.

I still don't know what the 2.712 TEC cooling problem is. I do know that it is not:

• the PWM controller.
• the Kp, Ki, or Kd values.

My next guess is that it has to do with this weird equation, which appears to convert temperature to volts? what? Yeah, I need to talk to Steven.

I currently need to talk to Steven about:

• soldering/wiring the NiChrome wire
• TEC cooling problem

4/4/11

Posted 17:13, 4 April 2011 (EDT)

I now know the reason for the background signal. The fiber optic cable is auto-fluorescing. BOO. It works okay for now (there is a small, but extant difference between H20 and SYBR+dsDNA), but in order to do any serious testing, I'll need to change the design. Or get a new fiber optic

It might, in fact, be time to get rid of the fiber optic all together. But I will also look around for fiber optics that really, really do not fluoresce.

Came up with a preliminary design for heated lid. Will verify with Steven.

v2.712 now uses thermistor equations instead of RTD. The correct temperature is produced. Hooray! However, The modeled temperature is quite wrong. More problematically, the TEC doesn't cool in this version (though it does in 2.52). This is probably due to the settings in 2.712 (i.e., it's comparing against the modeled temp instead of the real sample temp), as opposed to fundamental issues with the code. But I will talk to Steven about it.

4/1/11

Posted 15:50, 1 April 2011 (EDT)

Officially ordered 29-gauge wire.

The optics *do* detect a difference between water and SYBR dsDNA, but it's a fairly small difference. I think this small difference is due to the HUGE amount of background signal at the carrier frequency. Why is this happening? My current hypothesis is that the dichroic mirror is directly reflecting some light from the blue LED into the photodiode... but with the filters in place, this shouldn't be happening!

Question: how good are the filters we're using? Could they still be letting some other light through? (maybe we need another blue filter in front of the blue LED).

Steven went over with me some of the different existing versions of the LabView code. I will be working with versions 2.700 and later. Not all of them work. I will take notes on this; take a look at QRT-PCR:LabView-versions.

3/28/11

Turns out my calculations were all messed up before! With corrected calculations, and the data from [www.heatersplus.com], I decided on 29-gauge resistance wire for our heated lid.

3/22/11

Getting the temperature calculation right earned me a level-up... meaning I got to graduate to the Current Version of the Code. Got a brief intro to it. It's pretty complicated.

3/20/11

Based on model system (blue LED + ND filter), I don't think gain is high enough. Increased gain of transimpedance amp from 2.5e4 to 2.5e5. Well, it didn't help that much. So it's back the way it was. In any case, we get a significant signal (~0.25 units amplitude) with the model system. Huzzah!

After much wrestling with simple math, the temperature sensing is largely working! yay

3/18/11

Optical setup appears to be working.

3/16/11

Added dichroic mirror.

Question: how can I verify the optical setup? it seems to respond to driving signal, not fluorescence.

TODO: put away 25mm lens