Designing a Primer

Sequencing Instructions

1. 
   Design/check primers

• Want to sequence 200bp of protein coding sequence and 800 bp of vector if checking connection of protein coding sequence and vector

• Need forward and reverse primer for each plasmid to sequence beginning and end connections

• Check out OPRM1 visual of sequencing primers for an example in opioid project folder on the G drive

  • Pay attention to 5’ to 3’- the template strand may look flipped cause its in 5’ to 3’ order vs how it actually lines up with the coding strand (3’ to 5’)

  • Reverse primer for beginning and forward primer for end

B. Designing sequencing primers

1. Open the sequence file of gene of interest using ApE plasmid editor (blue and green loops icon on Start bar). The sequence can be found in Norimatsu Lab\DNA and RNA\Construct Sequences. You will see a list of folders for different proteins. Inside each of those folders, there are two additional folders: ORF and Plasmid. The ORF folder contains the open reading frame (coding sequence for the protein) Open the ORF folder and look for the sequence of interest.

2. Go to http://www.genscript.com/cgi-bin/tools/sequencing_primer_design. Copy and paste the sequence file. This designs primers for the whole ORF. If suitable primers are not found in the region of your interest, you can change the distance between primers.

3. Check for self-complementarity on the following website http://www.basic.northwestern.edu/biotools/oligocalc.html

4. Copy the desired primers into a Word document and save them for ordering.

• To order:

  • Use Genewiz

  • Click on oligo rapid synthesis.

  • Delivery format: Dry

  • Scale: 25 nmol

  • Credit card and shipping info already saved on website

2. Prepare primers

• If new primer add MBG water so that concentration is 250 μM

• Make aliquot with 1 μL of stock primer (250 μM) and 9 μL

• Aliquot should have concentration of 25 pmol/ μL

• NanoDrop aliquot to double check concentration

• Use pmol/μL to ng/μL excel doc to see what nanodrop concentration should be

  • Saved on G drive in inventory and database folder

• All other previously made aliquots should be 25 pmol/μL but you can nanodrop to double check

• NOTE: check nanodrop units- you want the concentration in ng/μL but lately the nanodrop has been measuring samples in μg/μL so you’ll need to multiply that number by 1000 to give you ng/μL in order to check the number from the conversion excel doc

3. Dilute plasmid DNA if needed

• Nanodrop to get the current concentration- see NOTE above about units

• Divide the concentration by

  • 500 if DNA length including the vector is <6 kb- usually will be this

  • 800 if DNA length including the vector is 6-10 kb

  • 1000 if DNA length including the vector is >10 kb

• The subtract 1 from the resulting number- this is how much MGB water to put in a separate aliquot tube

• Add that volume in μL and 1 μL of the stock plasmid DNA to make an aliquot diluted to 500 ng/μL

4. Prepare sequencing mixture

• Use PCR tubes

• Pull up Genewiz sequencing ordering on the internet

• Label the top AND the side of each PCR tube with the labels from the chart (ex. YN1)

• Add plasmid DNA, primer, and MBG water in correct volumes as specified above

• Total volume should be 15 μL

  • 1 μL of 500 ng/μL plasmid DNA (if plasmid length is <6 kb)

  • 1 μL of 25 μM(pmol/μL) primer

  • 13 μL MBG water

5. Order and ship

• Go to Genewiz and select sanger sequencing- plasmid

• Follow instructions

  • Service type: premix

  • Service priority: standard

  • Do not need to check save for prep

  • For primer info only need to put primer names under My Primer

  • Leave GENEWIZ Primer and Difficult template blank

• Place order- shipping and credit card info already saved

• Print order receipt

• Tape tubes in numerical order to 1st page using scotch tape- do NOT put tape over side labels. They will come off

• Get FedEx number from Dr. Norimatsu

• Take paper with tubes attached and FedEx number down to the mail room on the first floor- walk past the IT center and turn the right down the hallway near the end of the hall- mailroom will be on left

• Lady in the mail room will help you