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Primers & Sequencing

ELIM Biopharm: Primers & Sequencing

Designing Primers
Choose a forward and reverse primer from a location in the gene or plasmid that is sure to include the portion desired for amplification or sequencing For sequencing, it is desirable if possible to have primers that fall 50-150bp outside your desired region, to ensure that accurate reading occurs for the whole gene (often the first and last ~100bp in the read are very inaccurate). For PCR remember that the sequence portion corresponding to the primers themselves will be amplified also Primers should normally be between 15-30bp in length (around 20bp is ideal)

Desired melting temperatures are generally between 55-65°C As you will see, melting temperature is a function of length and GC content, so it is often difficult to design primers in regions much greater than 50% AT Forward and reverse primers should have the same melting temperature, or with a difference of no more than 3 degrees The annealing temperature used for a pair of primers should be set at 5 degrees below the lower melting point of the primer pair Using a tool like ApE or Geneious makes it easy to select certain sections of a sequence to check for primer features like melting point and GC content IDT's 'Oligo Analyzer' is a great tool to check for primer dimerization, hairpin structures, etc. Use this tool or something like it as a final check to make sure your primers will not be likely to react with themselves or each other around the temperatures they will be active for gene interaction. NCBI Primer Blast is another great tool. It can be used both to help design the primers and to ensure that the primers you choose will not amplify any genomic DNA in a colony based amplification. Primer Blast isn’t perfect. It will often miss off-target products, or predict ones that don’t happen. Special BioBrick considerations

Ordering Primers
We order our primers from ELIM Biopharm. The rest is fairly self-explanatory, but we'll do a walk through when you get here Primers <36bp ordered before 5pm will arrive the next day. Delivery is around 2:15pm.

Primer Dilution (stock preparation)
Once you receive your primers, you need to dilute them; Kosuke does 1/10 dilutions, but iGEM typically uses 1/20 (10μM) dilutions. Typically we create 100-200μl working stocks; it will take a long time to use up that much primer.

Example: 10μL primer stock
190μL qH2O or TE buffer

Sequencing
Two main reasons: (1) after a difficult pcr/gel extraction to ensure the product is correct or (2) after cloning/biobricking to ensure no errors were introduced during PCR.

Ordering Sequencing
You can put in the order using the same general procedure for ordering primers. The important difference is either before or right after ordering, you need to actually prepare the DNA that will be picked up for sequencing (see Premix Specifications below). For DNA pickups for sequencing, the guy usually shows up around 2PM, so if you want an order picked up day of, be sure to have everything put together by lunchtime. This can usually be done even if you're miniprepping the sample that morning; preparing the sample for sequencing doesn't take too long unless you have a lot of samples to prepare. He always picks up the samples from room 3, there's a file box labeled "ELIM." Print out the order confirmation sheet and staple a baggy with your samples to it and put it in the box. Premix Specifications (plasmid DNA). Prepare the DNA as specified by ELIM.

For plasmids, this looks like:
500ng DNA
0.8μl primer (one primer per sequencing reaction)
Top up w/ qH20 to 15μL

For sequencing other DNA (e.g. PCR product) see the ELIM website for specifications.

Checking the Data
Usually sequencing data will be available the morning after you put in the order, sometimes early, sometimes closer to lunchtime. The results can be accessed again through the ELIM site; after signing in, there is an option to "retrieve/download sequencing data." You can either just view, or download the files. I recommend downloading all the files because you'll want to view them all anyway. Tools like ApE or Geneious will be needed to properly read the files. Each sequence read will come with a '.ab1' file that visualizes the data, and a '.seq' file that actually gives you the sequence they read. Check the ab1 file first; you're hoping for strong clear peaks, where one of four different colors represents each possible base. Typically the beginning and end of the read will look sloppy, but the middle few hundred bases should look very pretty. If the read looks pretty clean, then open the .seq file and compare the sequence to the theoretical sequence.

      

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