9 replies to this topic

[neumanoid]

Hi,
I recently changed the thermocycler i was using to run my pcr's on from one that holds .65 microlitre tubes to one that holds .2 microlitre tubes and now i've lost one of my products. I'm doing everything the same as before i've only changed the size of the tube being used and the thermocycler.
Any help on why i'm losing one of my producst would be helpful.
Cheers!

[Adrian K]

Probably due to the material: the PCR tubes had different heat conductivity with your thermal cycler. This is what I can think of.

I used to get a specific sharp band in a thermocycler and when I change to other thermalcycler and different tubes, some unspecific appear. I had to re-adjust the conditions again and everything works as usual.

[neumanoid]

Reajust how though? i've never had to troubleshoot pcr conditions before. Should i make the elongation step longer? The annealing temp lower or time longer? Increase the denaturation time? Or all of the above till i get what i'm looking for?

[Prep!]

neumanoid, on Nov 16 2009, 07:13 AM, said:

Hi,
I recently changed the thermocycler i was using to run my pcr's on from one that holds .65 microlitre tubes to one that holds .2 microlitre

i ve never heard of 0.65 and 0.2 microlitre tubes (do u mean 0.65 and 0.2 mililiter tubes?)
Tubes shud not make a difference if they are of the same make or brand (eg polypropylene) although heat transfer can...
changing conditions shud be the last resort... check teh specifications of the PCR (like ramp rates etc) .. there must be some minor setting snag...
or just check the cooling and heating system of the cycler.... is it peltier vs circulated etc...

[HomeBrew]

Is the prior machine still available? What happens if you prepare a PCR reaction at twice the normal volume, aliquot one-half of it to each kind of tube, and run them, one in the new machine and one in the old?

If the old machine will accept the smaller tubes, you could go to three times the volume and put one large tube and one small tube in the old machine, and a second smaller tube in the new machine, and run them all.

[Prep!]

HomeBrew, on Nov 16 2009, 06:54 PM, said:

Is the prior machine still available? What happens if you prepare a PCR reaction at twice the normal volume, aliquot one-half of it to each kind of tube, and run them, one in the new machine and one in the old?

If the old machine will accept the smaller tubes, you could go to three times the volume and put one large tube and one small tube in the old machine, and a second smaller tube in the new machine, and run them all.

i fear if the cycler is for higher volume tubes.. the lower volume tube wont fit in!!! (there are different blocks for diff kinds of tubes)
the idea is good though... also if you keep it.. there will be air gap.. improper heat distribution!!

[gogreen]

HomeBrew, on Nov 16 2009, 06:54 PM, said:

Is the prior machine still available? What happens if you prepare a PCR reaction at twice the normal volume, aliquot one-half of it to each kind of tube, and run them, one in the new machine and one in the old?

If the old machine will accept the smaller tubes, you could go to three times the volume and put one large tube and one small tube in the old machine, and a second smaller tube in the new machine, and run them all.

I would go with Homebrew's suggestion...If the old PCR is available, that the best way to know what happens in these tubes is to run a reaction with all the variables, in this case, all supported different tubes in 2 makes of PCRs...its worth doing as you have a good lesson and a reference

[HomeBrew]

neumanoid, on Nov 15 2009, 07:43 PM, said:

...and now i've lost one of my products.

[emphasis added]


Also, the way you've worded this suggests that other PCRs are working in the new machine (and tubes), but one (in particular) isn't. This suggests to me that the PCR itself was originally iffy, and the new machine -- which may have some slight temperature calibration differences, different ramping rates, etc. -- might be causing this PCR to fail.

If other PCRs are working in the new machine (and with the smaller tubes) and just one reaction isn't, it's unlikely to be being caused by the tubes, but rather by the PCR experimental design of this one reaction being a bit marginal -- in terms of primer design or template secondary structure, for example -- and slight variation in the reaction conditions imparted by the new machine are the reason it's failing.

That's why I suggested what I did above -- you've jumped immediately to the tubes as the cause, but there are many other possibilities. Best to eliminate as many variables as you can, so you're doing as direct a comparison between the two situations as possible.

[klinmed]

neumanoid, on Nov 16 2009, 01:43 AM, said:

Hi,
I recently changed the thermocycler i was using to run my pcr's on from one that holds .65 microlitre tubes to one that holds .2 microlitre tubes and now i've lost one of my products. I'm doing everything the same as before i've only changed the size of the tube being used and the thermocycler.
Any help on why i'm losing one of my producst would be helpful.
Cheers!

The actual temperature of the pcr reaction mix always lags behind that of the heating block. Thus, the target temperature may not be reached in a larger volume of reaction mix (or a thicker walled tube). This is especially the case when comparatively short cycle times are used.

I would first try lowering the annealing temperature by a few degrees to compensate for the faster temp equilibration of the new tubes.

[ShannonJ]

You might also, just to be sure, find out what the ramping rate of the "old" thermal cycler was and see how that compared with the "new" one. Even for the same sized tubes I have seen ramping rates make a difference in products.