DNA Sequencing at UC Riverside Core Instrumentation Facility
1. Preparation of templates. Critical to successful
sequencing is the purity of template. Methods of purification giving good
template include the QIAprep series of products (from QIAGEN), including
individual columns, strips of 8 or 96-well plates. These silica membrane
procedures adsorb DNA in high-salt and, after washing, release it in water or
low-salt buffer. A second set of procedures employ Promega’s Wizard
products, also adsorptive silica, but attached to paramagnetic particles
instead of formed in membrane. Use of these products requires an additional
ethanol precipitation as a final cleanup. Useful guides to preparation of
template for sequencing reactions are available for download from Applied
Biosystems and Qiagen as part of larger reference manuals on automated DNA sequencing.
http://docs.appliedbiosystems.com/search.taf
(keyword search, Automated DNA Sequencing) and
http://www.qiagen.com/literature/handbooks/default.asp
(keyword search, DNA
Sequencing).
2. Submitting
templates for sequencing. Please download and print our login form
for use when bringing samples to CIF. Make sure that the sample name on the
tube cap matches that found in the first column of the login form; this greatly
reduces chances for confusion when we set up the sequencing reactions. In an
effort to improve service, we ask investigators to provide plasmid with insert
or PCR product and 3.2 pmoles primer in 12 µl water. This is twice the volume
we need for a single reaction, and it allows us to quickly do a second reaction
if the investigator and C.I.F. mutually decide the quality of the sequence read
is not up to standard. The quantities of template needed vary depending
on whether the template is a PCR product, plasmid with insert, BAC or cosmid,
or genomic DNA. For small numbers of samples, we prefer that
they be submitted in flat-top PCR tubes (e.g., from Phenix: cat. no.
S9-MPC-200F) so that the sample name can be written on the top with a
fine-tipped Sharpie. As already discussed, investigators may also include 3.2
pmol specific primer in 12 µl water.
For larger scale sequencing projects we accept templates in 96-well plates. This allows us to reduce our prices by automating the purification of extension products, and we pass this savings on to the investigator. Please use semi-skirted PCR plates with a single notch at upper right corner, e.g., Phenix MPS-3580 (we no longer accept Phenix MPX-PCR96-HS), and seal tops of plates. An appropriate seal is a thin adhesive film, e.g., Phenix LMT-THIN-EX. Our plate rates will also be honored for 48 templates filling the first 6 columns of a PCR plate.The quantities of template and primer and the volume of water are one half that requested for single samples, e.g., samples are submitted in 6 µl water instead of 12 µl. The naming of templates should include the investigator’s initials followed by a number incremented sequentially as additional templates are submitted from day to day. For instance, John Q. Scientist’s first sample is labeled JQS001. This scheme helps us avoid cumbersome notation and reduces the chance of error when we record sample names in the software used for data collection. As an alternative for those investigators, with large numbers of samples, wanting their unique identifiers to remain with the data files, please inquire about an option for sending an Excel file containing sample names by email to CIF; we provide a template spreadsheet by email that the investigator edits with their sample names and then returns to us. The first column of the spreadsheet should contain well identifiers from the plate, top-to-bottom and left-to-right (A1, B1,...H11, H12). In the second column please put your sample name. Send spreadsheet as an attachment by email to Yoon Gi Choi (ychoi@ucr.edu) on the day you bring the samples to CIF. We can then import your file into our data collection software, thereby partially automating sample name entry and eliminating the possibility of mistakes during manual entry.
3. Universal primers we keep on
hand and available for use with your templates (no charge).
T3 5’-AAT TAA CCC
TCA CTA AAG GG-3’
T7 5’-TAA TAC GAC
TCA CTA TAG GG-3’
M13F(-21) 5’-TGT AAA ACG ACG
GCC AGT-3’ (This will be used for M13F
without note)
M13R 5’-GAA ACA GCT ATG
ACC ATG AT-3’ (This will be used for
M13R without note)
SP6 5’-ATT TAG GTG
ACA CTA TAG-3’
Also available:
pTV 5’-TTT TTT TTT
TTT TTT TTT TTV-3’ (to overcome poly A in
template)
pAB 5’-AAA AAA AAA
AAA AAA AAA B-3’ (to overcome poly T in
template)
pCD 5’-CCC CCC CCC
CD-3’ (to overcome poly G in template)
pGH 5’-GGG GGG GGG
GH-3’ (to overcome poly C in template)
M13F(-40) 5’- GTT TTC CCA GTC ACG AC -3’
M13R(-24) 5’-AGG AAA CAG CTA
TGA CCA TG-3’ (For
pENTR)
pET-UP 5’-ATG CGT CCG GCG
TAG A-3’{For pET from Invitrogen (167~181) not from Novagen}
T7-TERM 5’-GCT AGT TAT TGC
TCA GCG G-3’ (For pET)
Note: "V”=A, C or G; "B"=T, C or G; "D"=A, T
or G; "H"=A, T or C.
4. Our DNA Sequencing PCR
conditions. Our standard reaction includes denaturation at 94° and
annealing step at 50° C and an extension step at 60° C. In some cases, i.e.,
with the use of higher melting temperature primers, it may be beneficial to
increase these temperatures or to exclude the annealing step in order to reduce
mis-priming. Investigators are invited to discuss these options with personnel
in Core Instrumentation Facility.
5. Retrieval of data.
Sequencing files are transferred to our server (I.P. # provided on signup).
Investigators are notified by e-mail when data is posted, and they can then
connect to our server in a secure environment with an ftp application to
retrieve their files. There are a number of free or low-cost ftp applications
for users with PCs; some of these used in CIF include Smart FTP, FTP Surfer and
Guild FTP. Users with Macintoshes may use "Fetch", available by
download from http://fetchsoftworks.com/;
the newest version costs $25, while an older version is free (save the S/N for
future free upgrades). A simple option for Mac users in OS X, which bypasses
the need for an ftp application, is to use the "Connect to Server"
option accessible under the "Go" menu of your hard drive. When the
connect window opens, type ftp://CIF server number (provided by CIF personnel),
then "Connect". After installing your ftp application or when using
the OS X connect feature, you will be given a user name and password by CIF
personnel (see Yoon Gi Choi for this). You will then gain protected access to
our server. It is sometimes necessary, as when using Fetch, to specify
“binary” type in your ftp application prior to download. Files,
complete with sequence text file and electropherogram, can be viewed on both
PCs and Macs (see below).
6. Viewing electropherograms and
text files on your computer.
For PC computers, an inexpensive application called
Chromas is available for download from this URL:
http://www.technelysium.com.au/chromas.html
A freeware application for viewing electropherograms (which can be converted
to text files), favored by CIF personnel, can be obained at: http://www.geospiza.com/finchtv/download/index.html.
Another free application called ABIView, for use in Windows, is available
for download from: http://bioinformatics.weizmann.ac.il/software/abiview/abiview.html
or http://www-sequence.stanford.edu/group/informatics/ABIview/
For Macintosh computers, it will be necessary to first
convert the Windows NT data files to a format that can be recognized by a
Mac application. The freeware, “Sample File Win to Mac,” is
available by download from Applied Biosystems: http://www.appliedbiosystems.com/support/download/3700/Conversion/ConvProg.sit
or by email from Tim Kingan (tkingan@ucr.edu). Place your files in a
folder on the Desktop and when prompted by the software for the location of the
files, "choose" but do not "open" the folder; files
will be converted in a second or so and can then be opened with Editview, which
is also available from Tim or by download from this URL: http://www.appliedbiosystems.com/support/download/SeqAnal/installs/mac/EditView_1.0.1.sea.hqx
or use FInchTV at http://www.geospiza.com/finchtv/download/index.html.
Upon download, both programs are self extracting files the install for which
can be executed by double clicking on the program icon.
7. Troubleshooting.There
are a number of situations that can lead to poor quality sequence data; these
situations are manifested in different and potentially diagnostic ways in the
appearance of electropherograms. CIF personnel are available to discuss
possible corrective action. A number of internet sites provide diagnostic
information and may help in pinpointing the source of problems. One such site,
with instructive examples is maintained at