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PCR Temperature change control malfunctioning

PCR

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#1 SoominP

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Posted 11 June 2014 - 09:54 PM

Hi, I'm a high school student who used PCR for a final AP Bio independent lab project.

My lab was about Alu DNA detection.

So I needed to amplify DNA sequences of PV92 on chromosome 16 using PCR machine.

The machine I used was Hybaid PCR Sprint produced by Thermo Electron Corporation which was merged into Thermo Fisher Scientific in 2006.

My teacher didn't have a manual but we could find one online published by Thermo Electron Corporation in 2002. 

My lab protocol (I used Edvotek's kit #333) required three different stages : Initial heating for 5 mins at 94'C, then 32 Cycles of 30 secs, at 94'C, 30 secs. at 61'C and 45 secs at 72'C, and final hold for 4 mins at 72'C.

So I programmed three different stages which are like this :

 Stage 1-step 1 : 5 mins, 94'C : 1 cycle

 Stage 2-step 1 : 30sec, 94'C / step 2 : 30sec, 61'C / step 3 : 45 sec, 72'C : 32 cycles

 Stage 3-step 1 : 4 mins, 72'C : 1cycle

And I did trial run by setting Stage 2 with just 1 cycle.

After a few minutes of pre-heating, PCR went through Stage 1 for 5 mins then entered Stage 2 and went through step 1 for 30secs.

Then it stopped and didn't proceed to step 2(61'C). The monitor showed gradual (very slow...) decrease in temperature of Block (0.2ml) and control Tube thermistor.

Though I was curious but hoped that it just takes some time in decreasing temperatures...

So I set my DNA samples(in 0,2ml thin tubes) and started actual PCR program and ran it overnight. 

Then, when I checked it this morning, it was again stuck at between Stage 2 step 1 and step 2. And the temperatures of Block and control Tube were not even changing (except for periodic changes of +- 0.1)....

I was so frustrated and felt hopeless and so did my teacher. He said he hadn't used that machine for years and couldn't find out what was wrong.

We think it was a mechanical problem, especially since that the lid of the machine couldn't be locked properly so I had to place a book (not so heavy,though) on the lid to hold it still....

But I'm still wondering whether there are some mistakes I made in setting the program or running it... &  whether it was actually mechanical error.

If it was a mechanical error, what can we do to fix it? I mean, is there any alternative way to run the program? or do we need expert's engineering helps?

Thank you so much for reading this and could you please give me some advice, if you have some?

Thanks!



#2 bob1

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Posted 12 June 2014 - 12:19 AM

Your program looks fine.  It may pay to check if there is a control for ramp rate (how fast the block is heated or cooled), it could be that it is switched off or set to very slow.

 

If there is an actual mechanical failure of the peltier unit (the bit that controls the heating/cooling) then it will be cheaper to replace the whole PCR machine rather than attempt to get it repaired.  There should be someone (an  electrician) around who specializes in lab equipment who can tell you if this is the case.  It could be the lid, but this would usually give a lid error (for most designs, I'm not familiar with your one specifically)







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