Sequencing Protocol

2015-01-13

azim58 - 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)

Don't add two primers (just one for sequencing and two for PCR)

Total volume: 6 uL

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: \\biofs\CIM\Administration\Administrative\Forms\DNA
Sequencing Form.doc)
CIM Cancer Account Number: FQ91002 (make sure to send Pattie form as
well) (old account was FQS0030)
DNA Sequencing Request form.doc
"C:\Users\kurtw_000\Documents\kurt\storage\CIM Research Folder\DR\2014\01-18-2014d1351\wikispaces download 01-18-2014d1351\01-18-2014d1452\DNA Sequencing Request form.doc"
DNA Sequencing Request form.pdf
"C:\Users\kurtw_000\Documents\kurt\storage\CIM Research Folder\DR\2014\01-18-2014d1351\wikispaces download 01-18-2014d1351\01-18-2014d1452\DNA Sequencing Request form.pdf"

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
[email protected] 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

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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

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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.

Questions

If a gel extracted piece is left over night, and then processed the next day, does it sequence well?
-It doesn't seem to sequence as well. I have an example of this on 5-30-14 of notebook and here (all the sequencing results were bad): "C:\Users\kurtw_000\Documents\kurt\storage\CIM Research Folder\DR\2014\06-04-2014d1602\DNA Sequences 06-04-2014d1547\Sequencing Results 06-04-2014d1555.xlsx"
Can a sample have a low nanodrop reading and still sequence well?
-Yes a sample can definitely have a low (even negative) nanodrop reading and still sequence well. On the other hand, some samples can have a high nanodrop reading and sequence poorly. I think the purity of the product is very important to the sequencing result.
--example of samples with low values for "Nanodrop reading", but then still acceptable values for "Length of good sequence" with good alignments.
---"C:\Users\kurtw_000\Documents\kurt\storage\CIM Research Folder\DR\2014\06-05-2014d1127\Sequencing results 06-05-2014d1126\Table of Sequencing Results 06-10-2014d1048.xlsx"
---"\\biofs.biodesign.asu.edu\CIM\Research\labdata\Felicia Craciunescu\cancer vaccine project 2013\frameshift pcr screen\6-10-14\Sequencing results 06-05-2014d1126\Table of Sequencing Results 06-10-2014d1048.xlsx"

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see also
Renaming DNA Sequencing Files
DNA Sequencing