I have developed RT PCR mastermix , now its required to add stabilizer in it? if yes which stabilizer we can use at which concentration.
Hi Maulik,
Good to have your own master mix rather than commercial. In general, glycerol, BSA, Triton-X 100, Betaine, DMSO, formamide etc are often used in conventional and real time PCR as an additive. I would like to know your buffer system whether it is KCL or Tris HCL. Based on the buffer system the concentration of additives or stabilizers will differ. However, above mentioned additives are widely used. Better, run the experiment with your basic buffer system and ingredients. Then, based on the result the choice of additive and concentration can be selected. As a general condition most of the buffer system contain BSA (1-10 mg/ml) and Glycerol (2-5%). Most of the amplifications occurs well in these concentration.
Hi Maulik,
Good to have your own master mix rather than commercial. In general, glycerol, BSA, Triton-X 100, Betaine, DMSO, formamide etc are often used in conventional and real time PCR as an additive. I would like to know your buffer system whether it is KCL or Tris HCL. Based on the buffer system the concentration of additives or stabilizers will differ. However, above mentioned additives are widely used. Better, run the experiment with your basic buffer system and ingredients. Then, based on the result the choice of additive and concentration can be selected. As a general condition most of the buffer system contain BSA (1-10 mg/ml) and Glycerol (2-5%). Most of the amplifications occurs well in these concentration.
- That's an unusual question but one I have expereince with:
- The most common component of any mix for long term stabilisation mix for preserving enzymes is 50% glycerol: The vicinal hydroxl groupings hydrogen bond the enzymes and stabilise them
- You then dilute that down to 10% in the working buffer which return the enzym to its optimal activity or Vmax: 50% glycerol will st5abilise the enzyme @ -20C or -80C and preserve its activity in the longer term but at 50% in effect the enzyme is in a straight jacket and so do not have maximal activity
- Also 5% BSA is a good idea
- In addition, make sure you add an effective buffer like HEPES (not Tris) to preserve pH over time and with freeze thawing
- For long term stabilisation of a master mix contaning reverse transcriptase there are other factors or excipient subtances that you could add to potentiate the effects of glycerol but you would have to empirically determine which works best: that is thaw aliquots at 3 months, 6 months and and 1 year and asay enzyme activity: This is what I had to do for my own mater mix with an enzyme in
- See attached paper
- If your master mix does not the actual active (protein principle) then adding glycerol BSA and Hepes buffer would be enough
Narayanan Kalyanaraman Thank you for your answer, my buffer base is tris HCl. I will try as per your suggestion and check. Thank you once again.
Laurence Stuart Dawkins-Hall Thank you for elaborate answer and explanation. It will help me a lot for further work.
Fine. Just follow this link to get an idea about PCR optimization. I always preferred this method to optimize. Most importantly, keep the record properly for longer time. I have my notes from 2008... The knowledge gathered while optimization will transform into wisdom after couple of year. The data will certainly help you to optimize and trouble shoot further experiments easily. All the best.
My opinion is better use components such as BSA (1-5gm/mL),, Glycerol (3-5%) and Tween 20 (0.05 to 0.01%) or Triton X 100 (whatever available) or DTT (usually 6.7 mM) as final concentration in your master mix.
We have few reports for influence of DMSO in reducing (by 20% or less) PCR efficiency. Hence, I usually consider it as last option.
Article A Simple Procedure For Optimizing The Polymerase Chain React...
I am not much sure of the efficacy of glycerol in RT-PCR, but glycerol is definitely a great stabilizer for preserving bacterial cultures. Thanks Laurence Stuart Dawkins-Hall
Just in case you were thinking of storage in dry state the use of trehalose sugars as stabilising agents is widely used. The attached is interesting regarding the use of these sugars
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