i have designed almost 5 single guide RNAs for CRISPR/cas12. After Gateway cloning, the possible PCR product should be almost 370bp and 4 single guide RNAs are giving the desired product. one single guide is giving almost 570bp product which is not required. i am using forward primer from cas12 backbone and reverse complement of the guide as reverse primer in all PCRs. what is the possible reason of the PCR product larger than required?
you can try to use any other forward primer from the backbone or even, can use gRNA's forward primer with reverse primer from the gateway vector. Such kind of size difference is also seen in the correct gRNA clones sometimes. If the difference using modified primers is not much bigger, you may even try to confirm that gateway by sequencing.
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