How to PCR amplify template to prepare gRNA and Cas9 mRNA

Preparing gRNA template for IVT without cloning

Forward primer:

5′- TGTAATACGACTCACTATAGGNNNNNNNNNNNNNNNNNNNNgttttagagctagaaatagc-3′

T7 promoter- Bold
Target site- N’s
crRNA- lower case letter (these sequences will bind to the plasmid)

Reverse primer :

5′-AGCACCGACTCGGTGCCACT-3′

Note: If you have 2Gs or 1G in your target sequence then you can leave out Gs from the T7 promoter. Having 2Gs at the end of the promoter will make transcription more efficient but  are not essential.

cas9 mRNA template generation for px330

Cas9 Forward primer (px330):

5′-TGTAATACGACTCACTATAGGGAGAATGGACTATAAGGACCACGAC-3′

Cas9 Reverse primer (px330):

5´-GCGAGCTCTAGGAATTCTTAC-3′

cas9 mRNA template generation for px335 (D10 nickase vector):

Cas9 Forward primer (px335):

5´-TGTAATACGACTCACTATAGGATGTACCCATACGATGTTCC-3′

Cas9 Reverse primer (px335):

5´-GCGAGCTCTAGGAATTCTTAG-3′

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One Response to "How to PCR amplify template to prepare gRNA and Cas9 mRNA"

  1. Shruti Chitransh says:

    While designing reverse primer for PCR amplification of Cas9 gene to transcribe it invitro, what specific points need to be considered so that while transfecting in living cells they produce active cas9 protein.

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