Lidstrom:Sequencing with GeneWiz
Return to Protocols
- Using the correct template mass and primer concentrations is crucial. If you submit less than the recommended ng of DNA, sequencing fails more often.
- If you have a low concentration plasmid prep, you can increase the mass you send by sending 0.5 uL of 100 uM primers (the stock concentration) instead of 5 uL of 5 uM primers.
- Janet recommends doing a multiple alignment in NCBI's Blast webpage. Do not use the APE program's aligner, especially if you have a repetitive sequence.
- If you get an imperfect match or fail to match to your expected sequence, you can loosen the stringency requirements by selecting "somewhat dissimilar" or "more dissimilar" instead of "highly similar."
- Always have good labels in your FASTA files to make sure you don't mix things up.
- If you are making variants of a construct, you can compare your sequencing results to all of the things you are making. First make a text document with each of the reference sequences you are creating by pasting them back to back in a text editor. Include descriptions for each e.g. "> SH3-FLS in pSB1A3". Paste your sequence from GeneWiz into the top box and all of the other sequences in the bottom box. When the results are returned, click the "max score" button & it will put the match with the highest score first. This is most likely your product, but a sequencing read can match to multiple designs depending on your project.