Designing and Resuspending Primers

Appendix A: Designing Mutation Primers

Designing mutation primers:

1. For Q5 Master Mix, go to nebasechanger.neb.com and click on the “Please enter a new sequence to begin” link to enter a template sequence. The following steps are for Q5. For PfuUltra Master Mix, use the online program PrimerX: www.bioinformatics.org/primerx

2. Open the desired DNA sequence which can be found in Norimatsu Lab\Inventory and Database\DNA and RNA\Construct Sequences. Highlight the entire sequence and copy and paste into the sequence window that pops up on the webpage. Name the sequence after the protein name and click continue.

3. The number in the mutant name is the original amino acid position within the protein. Amino acids are encoded by codons, which consist of 3 nucleotides each. To find the 3 nucleotide positions that constitute the codon of interest, multiply the amino acid position by 3. The product is the last nucleotide in the codon, so enter it into the box for “End” position.

4. Count back two numbers to get the “Start” position. The mutagenesis region will now be highlighted in the sequence box. You may have to scroll to find it.

5. The first letter of the primer name is the abbreviation for the original amino acid that we want to change. Check a codon table to ensure that the selected codon indeed codes for the original amino acid in the mutation name.

6. The last letter of the mutation name is the desired amino acid that we want to substitute into the sequence. Look up the corresponding codon(s) that code for this amino acid in the codon table. One at a time, enter each possible sequence into the “Desired Sequence” box and record each corresponding Ta (annealing temperatures) value that the website gives you. You may use an Excel spreadsheet for this.

7. Primers with the lowest Ta values are desired. When the ideal mutation is found, copy the table under “Required Primers”, and paste into Word document. Make sure the formatting doesn’t misalign the columns; use the tab key to adjust rows accordingly.

Designing and Ordering Primers

Overview

Primers are often shipped and received dry or in a lyophilized state. First create a master 100uM stock solution (1 for each primer) and then dilute into 10uM or 5uM working stock or aliquot. This reduces the number of freeze/thaw cycles the master primer stock goes through and reduces the chances of contaminating the primary source for the primer.

Procedure

1. Locate your primers in the package and ensure they are the primers you ordered.

2. Compare the nucleotide sequences and GC% on each primer label to the order invoice. The melting temperature will likely differ between the label and the order (this is not important).

3. Spin down the tubes using the centrifuge machine. SPIN ALL TUBES BEFORE OPENING! This is because dry pellet can often come dislodged during shipping and could be in the cap.

4.  Create a 100uM master stock by adding MBG water into the tube. Take the amount of nanomoles (nmols) on the label and multiply it by 10. That number is how many microliters of MBG water you'll add to the tube. 

Example: For 38.2 nmol of primer you would add 382uL of MBG water to make a 100uM stock solution. 

5. Label the top of each primer tube with its name and concentration (100uM).

6. Wait 10 minutes to allow the master stock to rest at room temperature and then mix well (centrifuge) before creating the working solutions.

7. Dilute the primer master stock in a sterile 0.6mL microcentrifuge tube. Add 1uL of master stock and 19uL of MBG water into the microcentrifuge tube. This makes 20uL of a 5uM solution. OR add 2uL of master stock and 18uL of MBG Water into the micocentrifuge tube to make 20uL of 10uM solution. If the primer is a sequencing primer dilute to 5uM. If it is a mutation or subcloning primer dilute to 10uM.

8. Label the aliquot tube with the primer name and concentration. Update the online corresponding Freezer C Box document.

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 (https://www.genewiz.com/en)

  • 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 OR change the unit setting on the nanodrop (there is a dropdown where it says the units in the top right corner in which you can change them to whatever you need)

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