Designing and Ordering Primers

Sequencing Instructions

1. 
   Designing primers

• Generally want to sequence between 200-600 bps, Tm ~60C +/- 5degrees. The optimal length is 20, but 18-22 is acceptable. 

  • 1. • If checking connection of protein coding sequence and vector: Only want your range to cover where they connect, the middle doesn't need to be sequenced. Start IN the protein coding sequence, move out to the vector. So, to get the beginning of the sequence, you need a REV primer, and to get the end of the sequence you need a FOR primer.

       • If checking a mutation: FOR and REV doesn't matter as much, you just want to get the point(s) you mutated.

• 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’)

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.

(Long story short: get the construct sequence from the google drive.)

2. Go to https://www.genscript.com/tools/dna-sequencing-primer-design?page_no=1&position_no=1&sensors=googlesearch . Copy and paste the sequence file. This designs primers for the whole ORF (plasmid). If suitable primers are not found in the region of your interest, you can change the length (bps) of your primers

3. Once you have found a good primer, copy/paste its sequence into the plasmid construct sequence to make sure there is only one match.

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

5. Copy the desired primers into a google document and save them for ordering.

• To order primers: 

  • Use Genewiz

  • Click on order --> oligo rapid synthesis.

  • Delivery format: Dry 

  • Scale: 25 nmol

  • Credit card and shipping info already saved on website

2. Prepare primers

• When you get the dry primer, spin it in the little centrifuge by the balance (Gretchen) before opening the tube.

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

  • • [(x nmol) (0.001)] / 250 = y * 10^-5 L  ---> y * 10 is the number of uL to add

    • Make aliquot with 1 μL of stock primer (250  μM) and 9 μL MBG H2O. Aliquot should have concentration of 25 pmol (pmol = uM x uL).  

• NanoDrop aliquot to double check concentration

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

  • Saved on Google 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 OR change the units on the nanodrop by tapping on the dropdown in the top right corner of the screen where it shows the current units. 

3. Dilute plasmid DNA if needed

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

Nanodrop instructions

•  Get the length of your plasmid in Kbp, multiply that by 10 and you have your desired concentration in ng/uL

ex) 7,600 bp plasmid ---> 7.6 Kbp ---> 7.6 x 10= 76 ---> desired concentration is 76ng/uL

• Dilute the plasmid to the desired concentration

  • • Might have to do a double dilution if the plasmid is too concentrated.

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. YN01)

• Add plasmid DNA, primer, and MBG water in correct volumes as specified in the Sample Submission Guidelines 

  • • Add 10uL plasmid at desired concentration (instructions above)

    • Add 5uL of 25pmol primer (instructions above)

      1. • Can also think of it as 5uL of 5uM. It means the same thing.

• Total volume should be 15 μL

5. Order and ship: Wait to ship until there are 10 samples, we get free shipping!

• Go to Genewiz and select sanger sequencing- plasmid

• Follow instructions

  • Service type: premix

  • Service priority: Priority

  • 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

• FedEx number: 633768110 

• 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

  • OR: take a left when you get off the elevator. Once the hall hits the stairs, take a right. Go to the end of that hall and the mail room will be on the left and the lady who can help you will be on the right. 

• Lady in the mail room (1st floor) will help you