9 replies to this topic

[dtae]

I am making a fairly complex homemade mastermix for qPCR (includes, KCl, betaine, MgCl2, Tris, flourescein, dNTPs, DTT, and SYBR Green I). I am thinking of making up a ton of it (without primers or Taq) and freezing aliquots at -4 for single use of each aliquot (no refreezing). I know I should light protect it because of the Sybr Green, flourescein and DTT. Are there any other things I should be concerned with or reasons I shouldn't do this?

Thanks,
Dan

[dtae]

I just came across a related topic on regular PCR which appears to be unresolved but is worth linking to nonetheless:
http://www.protocol-...h__1#entry32532

[dtae]

to continue talking to myself...making the mastermix and freezing aliquots has seemed to work ok so far. I don't add in either the primers or taq until I am just about to use it.

I spoke to another lab that consistently does this without a problem either.

D

[casandra]

dtae, on 03 September 2010 - 11:48 AM, said:

to continue talking to myself...making the mastermix and freezing aliquots has seemed to work ok so far. I don't add in either the primers or taq until I am just about to use it.

I spoke to another lab that consistently does this without a problem either.

D

....hi Dan, and why do you freeze them at -4? (just so this wouldn't be a complete monologue)....btw, in one lab I used to go to, we also froze homemade master mixes and there was never an issue about them but they were probably not as complex as yours...oh, and what's the fluorescein for?

Edited by casandra, 03 September 2010 - 12:03 PM.

[dtae]

it's actually closer to -15 last i checked..even though the thermometer in the freezer says between -4 and -7. it's the freezer we have...

D

casandra, on 03 September 2010 - 11:59 AM, said:

dtae, on 03 September 2010 - 11:48 AM, said:

to continue talking to myself...making the mastermix and freezing aliquots has seemed to work ok so far. I don't add in either the primers or taq until I am just about to use it.

I spoke to another lab that consistently does this without a problem either.

D

....hi Dan, and why do you freeze them at -4? Just so this wouldn't be a complete monologue....

[casandra]

...and now to continue my own monologue...ok, so you need to add fluoresceine if using the Bio-Rad cyclers bec the SybrGreen signal is not strong enough for the machine to calculate background levels for normalisation iow as a reference dye ....thanks for the info...and one last question, why such a concern for freezing your own master mix when most of the commercial mixes are stored frozen before use anyways?

Edited by casandra, 03 September 2010 - 12:53 PM.

[dtae]

I didn't have a particular reason to be concerned besides general paranoia gained from making mistakes that were obvious in retrospect. As for the fluoresceine, it seems to be very stable so I am not so worried about it--also I have a suspicion that the iCycler doesn't really need it despite the manufacturers recommendation.

Thanks,
D

[shirosands]

HI

I am going to start (trying) to do qPCR in my department, can I ask where do you buy the SYBR green from and how much it is?
I really don't want to buy a pre-made mix and be spending $1.80 per 50ul reaction  

I am looking into doing qRT-PCR as cheap as possible, and maybe ordering SYBR green to add to PCR mixes I already have.  

My department has a nearly 10 year old BioRad iCycler IQ machine.  Manuals for the machine recommend to use Mol Probes (now Invitrogen) S7567 which is a 10,000x dilution of SYBR green in DMSO.  Well that is for 1ml size which is $398 and now they have 500ul size, S7563 for $214 which is what I will buy if I don't find a better/cheaper source.  The manuals recommend to use this at 1:75,000 to 1:150,000 final dilution.  So I guess it would last a long time and be basically cost effective.  Sigma also sells a SYBR green 10000x dmso solution, 500ul for $283.  Both these solutions are recommended for use as gel stain but should also work in qPCR.  Invitrogen also sells SYBR (green I think) as a solid, 1mg for $186, item S21500.  A simple assumption is that the 10,000x dilution they sell is actually a w/v dilution of the solid, so 1mg in 10 ml (10,000ul) DMSO = 1:10,000x dilution (and gives you an idea of the typical product markup for these things).  So that would be even cheaper and would probably be more SYBR than I would need to use in my lifetime; has anyone tried that ?

ALSO !

Has anyone done qPCR with other nucleic acid dyes like Ethidium Bromide, Acridine Orange, or Propidium Iodide, (I prefer to use things I already have on hand and are much cheaper than SYBR green ofc!) etc. and if so what final concentrations to use them at please ?

Thanks a lot for any infos

[dtae]

It sounds like you are looking at the right SYBR Green (they market it as gel stain because they want you to buy their more expensive pre-made mixes). I think ABI owns the rights to it and other resellers might just be buying it from ABI. Not sure about re-suspending yourself--i guess it should work fine, but haven't fooled around with that.

I've had some problems with the iCycler in terms of well position effects (I have another recent post on this--just search for it) - so be careful. I would recommend avoiding using corner wells at least..and while validating things, try to stay in center wells and keep your well positions the same.

I think I've heard that Ethidium Bromide is no good for qPCR. I forget why, but I vaguely recall that it is a potent PCR inhibitor at concentrations where it would fluoresce enough to be detected. I don't know about the other stains--but I would avoid experimenting and stick with SYBR, unless your time isn't that valuable and you like such experiments. But please report further if you find out anything.

Best,
Dan

[Trof]

Just an idea..
you can run 20ul reactions instead of 50ul if your cycler suports it (and it does not evaporate), that saves cost too.