I am currently working on in vitro transcription and got the problem with low mRNA yield.
I prepared fresh IVT buffer and 2.5ug DNA template for 100ul Rxn and used N1-Methylpseudo-UTP, ATP, CTP, and GTP (5ul for each) with CleanCap AG (4ul) for 3 hours incubation in the thermal cycler. And then purify mRNA by RNeasy Mini Kit (QIAGEN. 74104). However, the yield is only 30ug per 100ul Rxn which is quietly lower than the expected yield on the protocol. The mRNA length is about 3K, and I make sure the DNA template is followed as CleanCap AG design (with AGG).
I used the T7 RNA polymerase (New England BioLabs cat. no. M0251S), and the recipe of the 10X buffer as recommended by the Trilink protocol.
Hi,
Have you checked the RNA on an agarose gel electrophoresis? If so then you can use it for the RT-PCR. Else you can try increasing the DNA template.
the yield of invitro transcription varies from one template to another. check if the length of your RNA product is OK by agarose electrophoresis (use a clean agarose gel aparatus, denature your RNA before loading...). If the RNA is correct (not degraded) then try to incubate for a longer time, add more DNA template.
Actually, I checked the RNA size which was correct, and I tried to add DNA template up to 4ug. However, the yield was still low.
Maybe I will try another method for purification.
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