In vitro Transcription (#IVT)is one of the complex and costly reactions in #MolecularBiology and at the same time pretty versatile and robust system to produce patient-ready #therapies. So there is often a lot of regulatory criteria to be met. Hope the listed suggestions help you meet them!
A) Inorganic #Pyrophosphatase addition: #Pyrophosphatase serves two purposes:
It cleaves the pyrophosphates formed, as the #transcription reaction progresses. By following #LeChateliers principle, the position of #ReactionEquilibrium then shifts to counteract the change and to reestablish the equilibrium in favor of more transcription where the production of #RNA increases.
Secondly Pyrophosphates in the reaction mix coordinates with #Mg2+ and precipitate out. Since #Mg2+ is a vital #cofactor for #RNApolymerase activity, its precipitation will reduce reaction efficiency, and thus #Pyrophosphatase addition comes into picture. The recommended dose is 0.002U per uL of reaction mix.
B) #Spermidine addition: Spermidine [N-(3-aminopropyl)- 1,4- butanediamine, C7H19N3] is an inexpensive polyamine additive used a large array of restriction digestion, #PCR and IVT reactions. It is used for the following reasons:
#Polycationic compounds like these interact better with the oppositely charged nucleic acids than Ca2+ and Mg2+ ions where charges are punctual, thereby promoting condensation and aggregation of #DNA.
These molecules stimulate #RNA transcription with DNA dependent #RNApolymerases from #phages.
C) #Dithiothreitol (DTT) addition: #DTT is a reducing agent used in virtually all reaction buffers to increase the life of enzymes containing free sulfhydryl groups in a reaction tube, limiting the natural #oxidation that would inactivate them.
D) #Triton X-100 addition: #Detergents like Triton X-100 and #Tween20 are added in reaction mixes to reduce adsorption to the plastic tubes.
E) #PEG-8000 addition: PEGs are usually added as #CrowdingAgents to improve the #efficiency of the reaction by increasing local concentrations of each reagent.
F) #MnCl2 addition: MnCl2 serves two causes:
G) #MgCl2 addition: #Magnesium is one of the most important ion in the #IVT reaction and is included by default in all types of #IVT buffers. But the trick is that its concentration should be in 1.1875-1.75: 1 ratio with the total #NTPs.
H) #RNaseInhibitor addition: Commercially available #RNaseInhibitors are known to be recombinant acidic proteins of about 50kDa with a #leucine-rich repeat. The #affinity of #RI for RNases is among the highest for any bi-molecular interactions and the dissociation constant of the RI-RNAse A complex is in the fM range under physiological conditions. This is added in #IVT reactions to protect the nascent #RNA being produced. The recommended dose is 1U per uL of reaction mix.
More to dig into:
Why magnesium chloride and not magnesium acetate?