Sequencing+Protocol

Sequencing Protocol

5 uL Sample (total of about 120 ng plasmid diluted in H20) 1 uL of 20 ng/uL primer (this can be prepared by diluting 10 uM primer by 4) (alternatively just add 1 uL of 10 uM forward or reverse primer for a final of 2.5 uM) Total volume: 6 uL
 * Don't add two primers (just one for sequencing and two for PCR)

Sequencing Form (for Life Science Sequence Lab (ASU School of Life Sciences DNA Laboratory); Josh Lebaer's lab also has a sequencing facility that can be used (DNAsu: http://dnasu.asu.edu/DNASU/)) (last seen: Sequencing Form.doc) CIM Cancer Account Number: FQS0030

Accessing files If the lab needs to send a lot of files, they may put the results in a folder rather than choosing to e-mailing the files. Here are the instructions from them.

8-16-12 To access the site go to ftp://dnalab.asu.edu Your user name is kurt@dnalab.asu.edu and your password is 123kurt. You should find this preferable to email but if you have a problem accessing the folder, let us know. Internet Explorer usually works best. Be sure to enable FTP folder view in the advanced options of Explorer. Only you and those you wish to have the password will have access to the folder. You may also download a free program called Filezilla. It accesses the FTP site very easily. http://files.uberdownloads.com/software/ftp-clients/filezilla. html

Designing sequencing primers: primers should be 50-100 bp upstream of the site to be sequenced. Typical sequence results are about 800 bp long. Notes about designing primers

Evaluating sequencing results If you have a good sequence, but it is very short, then you probably did not have enough template. If the sequence is very poor, then you probably did not have enough primer.

see also Renaming DNA Sequencing Files DNA Sequencing