Cas9 cassette for Transient CRISPR method

I am trying to amplify Cas9 cassette in pV1093.Sometimes I see the sharp band on gel after running PCR and sometimes not.Most of the times it is the probability case.The length of band is around 7kb and I use Phusion enzyme Annealing temp of 63 degrees and extension of 5min30sec with final extension of 10 minutes and 34 cycles.

After seeing the band I use ethanol precipitation to retrieve my DNA.Is there any other method?

Making Cas9 is really time consuming if not getting it amplified rightly on time.

Any suggestions will be highly appreciated.


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Orchid Gnomeover 3 years ago

Are you doing in vitro transcription? Just linearize it towards the 3’ end instead of PCR amplify it.

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