I received a plasmid from a research group and suppose to make RNA from it for transfection. The plasmid has a T7 promoter, I also noted that it needs "A cap" instead of the more common "G Cap" at the 5'end. I am using MEGAscript T7 Transcription Kit to make RNA from linearized plasmid. After many attempts I am unable to transcribe the DNA, the kit positive control is perfectly working. I tried to troubleshoot, which are as follows:
1. Checked the plasmid by restriction digestion
2. Linearized plasmid and purified by both column purification and phenol
chloroform method and used it for in-vitro transcription independently
3. Kit positive control is working.
4. First I used G-cap couple of times but later on figured out it needs A cap. It seems using A cap is also not working.
Can any one help me on this....
When you say it doesn't work, you are getting nothing or less than the positive control from the kit? Templates will have a varying efficiency/yield depending on other elements in the 5UTR.
Did you sequence? And check the T7 promoter? Furthermore is the T7 promoter in the right orientation or used for expressing another ORF?
Did you sequence the plasmid to check the T7 promoter is correct and in the correct orientation for the particularly ORF on the plasmid you want?
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