Adding G at start of sgRNA oligo's

[d.ker...@ed.ac.uk]

Hi,

 

I'm using vectors PX330 and PX335 for my CRISPR experiments. When designing the sgRNA oligo's you mention that adding a G at the start of the sgRNA oligo sequence helps with trasnscription initiation by the U6 promoter. I used the CRISPR tool to design my primers and chose the top 6 highest score primers for my experiment. Some of these were  primers for the reverse strand, so does that mean I add the G to the the 5' end of the reverse primer or to the 5' end of the forward primer?

 

For example :

 

CRISPR tool designed primer 5'-TAGTGGCGAGCGTGTTCTCT-3'for the reverse strand.

 

So to clone into BbsI digested PX330 add AAAC  5'-AAACTAGTGGCGAGCGTGTTCTCT-3'

 

the compliment primer on the forward strand would be 5'-AGAGAACACGCTCTCCACTA-3'

 

Add CACC to become 5'-CACCAGAGAACACGCTCTCCACTA-3'     5'-CACCAGAGAACACGCTCTCCACTA-3'

                                                          3'-TCTCTTGTGCGAGAGGTGATCAAA-5                

 

 

Thanks in advance

 

David

[julia]

Hi Davia,

     I think you've mistaken this problem. The gRNA you designed is used to add to the vector. And you are right that a G at the start of the sgRNA oligo sequence helps with trasnscription initiation by the U6 promoter. Whether the gRNA target to the forward or the reverse strand has nothing to do with the G after transcription. I hope that will help.

best regards

 Julia

在 2014年2月19日星期三UTC+8上午12时26分18秒，d.ker...@ed.ac.uk写道：