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Created page with "#Go to http://www.ncbi.nlm.nih.gov/nuccore/ and find the sequence of interest. #Scroll down to the sequence and highlight/copy the nucleotides corresponding with your CrispR ..."
#Go to http://www.ncbi.nlm.nih.gov/nuccore/ and find the sequence of interest.
#Scroll down to the sequence and highlight/copy the nucleotides corresponding with your CrispR cut site.
#Go to http://www.ncbi.nlm.nih.gov/gene/ and find your gene. Paste your cut sequence into the ‘Find’ box. A new dialog box will appear. Click on the ‘sequence’ tab and select your sequence. The CrispR cut site will appear highlighted on the gene map.
#Zoom out to where the visible sites flanking your cut site are an appropriate size for primer design (~70-150 bp).
#From the ‘Tools’ tab, select ‘BLAST and primer search’, followed by ‘Primer BLAST- visible range’. A new tab will open. In this new tab, scroll down and click ‘Get Primers’. Primer BLAST will run automatically.
#When the search results appear, paste your cut sequence into the ‘Find’ box, just like in step 3. You will be able to see which primer pairs flank your cut sequence and which do not. Based on these options you should be able to select a primer pair that does not overlap with your cut sequence.
#Scroll down to the sequence and highlight/copy the nucleotides corresponding with your CrispR cut site.
#Go to http://www.ncbi.nlm.nih.gov/gene/ and find your gene. Paste your cut sequence into the ‘Find’ box. A new dialog box will appear. Click on the ‘sequence’ tab and select your sequence. The CrispR cut site will appear highlighted on the gene map.
#Zoom out to where the visible sites flanking your cut site are an appropriate size for primer design (~70-150 bp).
#From the ‘Tools’ tab, select ‘BLAST and primer search’, followed by ‘Primer BLAST- visible range’. A new tab will open. In this new tab, scroll down and click ‘Get Primers’. Primer BLAST will run automatically.
#When the search results appear, paste your cut sequence into the ‘Find’ box, just like in step 3. You will be able to see which primer pairs flank your cut sequence and which do not. Based on these options you should be able to select a primer pair that does not overlap with your cut sequence.