Automated Sequencing Service

Plant Biotechnology and Genome Center

Automated Sequencing Service

Welcome to the automated DNA sequencing facility

First time user's, please contact:

Dr. D.A.Lightfoot	Jawad Afzal
618- 453-1797	618-453-3121
ga4082@siu.edu	ajafzal@siu.edu

For Technical questions and Sequence appointments, Please Contact:

Samreen Kazi
618-201-1932(mobile)
618-453-5727(lab)

All prices indicated in this page are SIU intern prices. For the outside users, the genome center offers sequencing services at extra charge. For more information, please contact:

Dr. D. A. Lightfoot
618-453-1797
ga4082@siu.edu

Services Available

call

Preparation of DNA Template:

DNA quality and quantity are extremely important in automated sequencing. Cesium- banding methods, alkaline lysis/PEG precipitation and QIAGEN DNA isolation procedure provide sequencing quality DNA. We suggest that you quantify your DNA with a flourometer and run it on gel to verify concentration and purity. Resuspend DNA in water only.

Suggested Purification Methods with QIAGEN kits:

Plasmid DNA	QIAGEN Spin mini kit#(27104 (50 prep) or#27106 (250 prep) QIAGEN Plasmid Mini kit #12123 (25 preps)
PCR Product	QIAGEN Gel Extraction Kit #28704 (50 preps)

Alkaline lysis protocol can be provided on request.

QIAGEN products are available at the automated DNA sequencing facility. See Rubina Ahsan for a product list and save shipping and handling and 5% discount.

The ABI kits are available from applied Biosystems (800) 327-3002.

This will determine the cycle sequencing program used to incorporate the fluorescent dideoxynucleotides into extension products. Each kit basically comes with Taq DNA polymerase, fluorescent-ddNTPs, & dNTPs. There are four different fluorescent dyes that distinguish between the four bases in DNA. The reaction is linear, only one primer is used per reaction.

Soybean Genomics Center remains open from
8:00 AM to 5:30 PM Monday - Friday.

Other Suggestions

1.	Loading buffer will be provided to the customers (free), if someone wanted to make its own loading buffer, they must follow our protocol for automated sequencing loading buffer.
2.	Sephadex columns can be provided on charge.
3.	Users should provide at least 10 samples to make a run.

Notes To Sequencing Clients:

We try to have results within 72 hours after the samples are received. If you hear nothing within 5 working days, please call or e-mail us.
To speed the processing of your samples, please assist by observing the following items;

WHAT TO BRING:

1.	On campus users, please bring a purchase order number, and the name, address, and phone number of your accounts payable person or department.
2.	Submit primers and DNA in 1.5 ml tubes.
3.	Label the top and side of your tubes with some form of identification: initials, sample name, etc.
4.	Use label tape, but position any tape on the tube so that the tube will still fit into a microfuge. Do not overlap tape. Self-stick labels come off. Use black sharpie markers, not other colors. Do not scotch tape over hand written labels. We need to remove the tape and it often peels the label or writing.
5.	Maintain a similar concentration around 0,25ug/ul to 30ug/ul for all templates, which you submit.
6.	Please resuspend your samples in dH2O, not TE.
7.	Submits extra template. Reactions sometimes need to be repeated for reason beyond our control. For 1 reaction please send enough for 4 reactions. If extra template is not submitted with the order, reactions can not be tried a second time. Loading buffer will be provided to the customers (free), if someone wanted to make its own loading buffer, they must follow our protocol for automated sequencing loading buffer. Sephadex columns can be provided on charge.
8.	Users should provide at least 10 samples to make a run.
9.	Submit PCR products in dH2o at a concentration 1 or 2ul are used per reaction. 5ng/100 bases are needed per reaction. I.e. for a 600 base pair PCR product, 15-30 ng/ul.
10.	Limits your sample name to six characters or less. Periods, slashes and dashes are character. The simpler the name the less likely we will confuse samples.
11	Submit the entire same template in one tube.
12.	Submit primers already diluted to 5pmole/ul. Please label as pmole/ul. Do not dilute primers in TE. If you have degenerate primers, which we do not recommend, then submit primers at double the concentration for each degeneracy. Limit your primer names to five characters or less.
13.	Keep the TM of your primers as determined by oligo 4 or 5 between52-58oC whenever possible. We use thermal cycle sequencing and anneal at 50oC for 10 seconds. Avoid primers with very high TM (Over 65oC) which may anneal secondarily. Do not make primers with TM below 50oC. Calculate the TM of your primer. One of several methods used for calculating TM is; TM= [(A+T) x 2oC] + [(G+C) x 4o C] -5oC
14.	Clean template, free of salts, accurately quantified will improve your results. We can provide various procedures for template preparation. Thank you for following these guidelines, together we can get good results.
15.	Please label your pre-formatted 3.5" floppy disk with the same name as the person submitting the samples if you would like to receive your data on a disk. Submit only 3.5" disks. Do not submit 5.25" disks. (Zip drive option is available)
16.	If you are a Mac user, we encourage you to use FETCH to retrieve data electronically. Note we do not e-mail sequences. Please supply a user ID and password (no more than 8 character, all lower case preferable) for data retrieval.
17.	A technician will prepare users disks or files at one time after the raw data has been transferred to process data. If there is no disk, a folder will be left on the desktop of the computer for 1 week, all the files can be retrieved via FTP, A password will be assigned to the each user of the facility to retrieve his or her sequences.

SEQUENCING RESULTS AND ANALYSIS

Base read of 500-800 bp is possible when template are prepared properly. Sequence data results have a printed color electropherogram. Electronic data in simple text and an electropherogram format. These results are available the morning following the gel run at the latest.