Hi everybody,
I have tried to make my home-made master mix for our laboratory. I have used two type of dyes , cresol red and Bromophenol Blue(BPB). I see when I use BPB my PCR is inhibited but no inhibition is observed for cresol red.
Have anyone had the same experience? Do you think the pH of BPB need to be adjusted before use? when I add BPB to my colourless master mix in the proper concentraion it return to blue so I think pH readjustment of master mix buffer is not needed. How do you think ?
Hi Hari Shankar Gadri ,
Acutally I am going to make my home made-master mix and then want to make it commercialized. Therefore, it need to be colourful. I chose bromophenol blue for this aim but I see than it inhibits my PCR.
First I dissolved the Bromophenol blue into DW in a proper concentration based on the protocol https://www.protocols.io/view/preparing-1x-pcr-master-mix-ceqhtdt6.pdf. The colour was still redish so I could see that it's pH was still acedic. when I added this solution to my prepared master mix it returned blue. But when I move forward to PCR my reaction had been inhibted while my control master mix ( without dye) had no inhibition.
Do you think it might be because I did not filtered the prepared bromophenol blue solution or I did not adjust the pH before adding it to Master mix?
Hello. you can Experiment with different concentrations of BPB in your master mix to see if there's a concentration at which PCR inhibition is minimized, Check the purity of your BPB stock solution and ensure it's free from contaminants, and Investigate whether there are any known interactions between BPB and PCR components, and adjusting your protocol accordingly.
What's the market share you're hoping for? There are a LOT of existing "ready to use" PCR mixes that include a colorful dye. And they are cheap due to industrial scale manufacturing. If you already have one dye that you know doesn't inhibit PCR, why are you trying to make this work with BPB?
Dear Katie A S Burnette ,
We are have own customer and about to lunch our industry. So we should not be dependent on other's. Therefoe we have to formulate our Master mix from the basic. Also, by this, we want to attract them to our other products, since master mixss are commonely used in any laboratory.
Dear Mahnoosh Maleki ,
Thank you for your response,
Actually we bought the BPB product from one of the Biobasic distributor in Iran. I checked the Biobasic Co. website. there is not information about the purtity of the product. Based on my inverstigations, BPB might has inhibtion effect on PCR in high concentraion I found little source which confirm this. But if you look at common master mixes you see BPB is commonely uses for this aim. Furhtermore, I dont think the pH adjustment is needed for BPB solution prior to use. Maybe next time I will try to prepare it in Tris buffer and avoid vortexing it to minimize the carry over of the probable impurities.
Katie A S Burnette Mahnoosh Maleki Hari Shankar Gadri
Thank you for your great response. Actually I found the issue source. I was trying to amplify a hard GC reach segment with the new developed Master mix. I found out that Trehalose concnetration in my new developed master mix shifted the Tm of the primers by sevral degrees. I decresed the Trehalose concentraiton to 0.1 M which now it works very well.
Best wishes.
Ali
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