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