For my promoter activity analysis, I take 0.12pmol of each different size of construct and 1:100 ratio of renilla vector. I balance the amount of total vector using a filler plasmid.
1) Construct 1= Firefly 500ng + Renilla 5ng+ Filler 5ng ( Total 510ng)
2) Construct 2= Firefly 480ng + Renilla 4.8ng+ Filler 25.2ng ( Total 510ng)
Because I maintain 1:100 ratio, so depending on my firefly , I change the renilla amount. Some papers, I observed that although they maintain equal molar of firefly plasmids of different sizes, but take same amount of renilla plasmid, not same ratio. Which one is correct- same amount or same ratio?
Looking forward for your reply
Thank you
Hi Mayank,
You mixed two different methods of calculation in your experiment which in my view is not correct. I will explain in brief:
1. You took good decision of taking equal moles of your construct. This reduced your chance of artifact since you took equal number of DNA molecules of different construct.
2. Instead of maintaining the molar ratio you used mass as a factor to maintain the 1:100 ratio of Renilla luciferase, which is wrong. As you can see in your equation, you took different pmoles of renilla in different subsets of your experiment that will introduce error in your analysis.
My recommendation: based on the molecular weight of Renilla luciferase take equal pmoles of this in all your subsets keeping the 1:100 molar ratio with your constructs (rather than ratio of their masses).
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