Many commercial diagnostic kits send single reagent (Premix) to which the user must just add respective DNA and perform Real Time PCR.
What are the key parameters to be considered while preparing such premix which would have (Taq enzyme, primers, probes, Taq buffer)
Note: Consider that the primers, probes and taq enzyme concentrations are already standardized for a target (when individual constituents are added seperately)
Please share experiences, related articles or references in development of such premix
I didnt get your point exactly.
When you use a premix the guide of that explain that you have to add how much DNA, specificity and sensitivity of primers they used, range of ct or etc.
Hello Ali
the motto is to develop a pre-mix (with all necessary primers, probe, taq buffer, taq) for a specific set of SNP's. This will allow the end user to take say 10 ul of premix and 5 ul of DNA for a 15 ul reaction. So that every time a set of samples are to be analyzed one need not add primers, probes and other reagents seperately.
In such case
My question is if I have to develop my own premix, where in I add primers, probes, taq enzyme, buffer for say 1000 reactions, are there any factors involved in storing such premix as a whole and use it.! does that effect the primers, probes or taq enzyme shelf life.
there are few kits who use premix (with taq buffer, primers and probes) as one consitutent and Test DNA to be added along with Taq enzyme alone seperately
others provide premix (with all pcr constitutents) and user have to add DNA alone per reaction.
I hope u got it now ?
I see.
Most of premix that I used was for a simple PCR.
I think in rqPCR so you can prepare a premix but you have to considering the highly sensitivity of each primers, probes and etc.
I think you can prepare 1000 reactions for example and then drive it to avoiding freez and thow them to less damage.
one problem is to avoid primer dimer so if I were doing this I would add a hot start enzyme so there is no enzyme activity prior to starting cycling. Trehalose sugars are also very good at stabilising enzymes and reagents for very long term storage particularly if you were thinking of storing the mix lyophilised
Hello Paul that was a very valuable input. I would consider using Trehalose a try.
Hello Paul
Talking about Trehalose in maintaining the thermostability of Taq, I did go through few journals. they claim exposure of taq enzyme to 95 C till 90 minutes did not loose its enzyme activity in the presence of 0.2mol/L trehalose.
But can we co relate it to long term exposure of enzyme at temperatures beyond 50 C for 6-10 days. Like do you think addition of Trehalose could improve the thermal stability during long term storage at temperatures 45-60 degrees. (The purpose is, we are facing stability issues while shipping our taq master mix), does addition of trehalose sort this issue.
hello Gautham,
I think that these sugars protect long term biological activity at intermediate temperatures as I think that some small fish use them to protact their cells when small ponds dry out and the fish remain dry and alive until the next rains revive them.
One aspect I forgot is that for increased stability most enzymes are shipped in higher glycerol than the reaction glycerol concentration but glycerol is slightly denaturing and can effect the binding specificity of dna primers so the pre mix will need to be tested for the higher glycerol concentration in the reaction mix if you do decide on raised glycerol and liquid shipping.
I am not surprise that taq keeps its activity at high temperatures.It is very stable and some companies used to ship taq at ambient temperatures because it was so stable and only stopped because of customer resistance. I think that the main risk of warm shipping would be bacterial contamination growing and not enzyme degradation. Good quality NTPs will probably help in making up stable mastermixes as well
good luck
{aul
6th Jun, 2018
Goutham Nageshwar
AURA Biotechnologies Pvt. Ltd
Paul, NTPs point does seems to be another valuable input, any idea about stability of NTPs,? sending at ambient temperatures will it effect NTPs degradation?
We do generally ship using Gel packs (which maintain 4C for 1-2 days)
Coz we once sent Taq Enzyme alone to one of our clients at ambient temperature which took more than 6 days to reach them, but it did work excellently.
The same enzyme when used for a master mix and after 6 days of ambient temperature no activity at clients end. Could it be coz of NTPs quality here?
6th Jun, 2018
Paul Rutland
University College London
I think the problem with NTPs is hydrolysis to NDP which are chain terminating so inhibit pcr , Lithium salts , not sodium, are more stable and this effect is most noticeable on repeated freeze thawing but it should be worth testing Li salts if you do not already use them routinely as they also offer resistance to bacterial degradation
this address has a little information
https://www.bioline.com/sg/dntp-mix.html
7th Jul, 2018
Vikram Kumar
National Centre For Cell Science, Pune
Hi Gautham,
You can store all them together as Paul suggested. In my experience, We prepare master mix for qPCR. Even, many a time, we store them at 4 degree for a day. We make our own SYBR green mixture. Only thing you need to consider is that use hot start polymerase e.g. Jump START POLYMERASE.
Another, example of such kind of kits are: miRNA profiling kits. e.g QuantiMir kit for miRNA expression analysis. Just go through there storage and shipment protocol. It might help you.
7th Jul, 2018
Goutham Nageshwar
AURA Biotechnologies Pvt. Ltd
Hi Vikram bhai.,
Yes for now we donot have a hot start, we need taq specific anti body for it, but we are looking into utilization of BSA and Trehalose in improving the thermal stability of our enzyme and master mix, and yes we are also considering lithium chloride ddNTPs
But most of them are suggeting there would not be much difference between Na salt Ntps and Li salt Ntps.
I shall write back for further clarifications
Thank you paul
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