Organisation of the sequencing facility at the Department of Genetics

• Tasks of the user
• Tasks of the facility
• Contact persons

We are running an ABI 3730 sequencer with 48 capillaries and automatic loading capacity from up to two 96 well plates.

The current service encompasses only the running of sequence reactions that are provided by the user. We plan to extend this into a full sequencing service in the future. Please check this web site for further updates.

The charges will be calculated on the basis of the expenses for the capillaries and general chemicals. We expect that they will be in the order of 0.50 Euro per sequence.

Current procedure

Tasks of the user

• preparation of template DNA
• run sequencing reaction using ABI cycle sequencing kit
• label reactions according to the general code
• deliver sequencing reaction in 8-tube strips in a volume of 20 microliter to sequencer room at Zuelpichertsrasse ground floor (room 032/033) until 10am each morning
• collect sequencing results from the folder via the network (usually next morning)
• convert into Mac format if necessary

Tasks of the facility

• the sequencing reactions are recorded in the daily processing sheet
• the reactions are purified on sephadex spin columns in a 96 well format
• the reactions are recovered in microtiter plates with 10 microliter loading buffer per plate
• up to two microtiterplates are loaded into the sequencer
• the sequencer runs from approx. 12 noon until next morning
• the runs are checked in the morning and the results are transferred into the folders that can be accessed by the users via the network

Contact persons

Karin Otto (tel. 4605) runs the service, but she will occasionally be substituted by other technicians.

any general problems should be reported to Diethard Tautz.

[Sequencing kit]

You can still use BigDye version 1 or 2, but you have to indicate this on sample delivery. Each particular run of 16 samples has to be adjusted to the correct BigDye version.

[Template DNA]

Note that the quality of the template is usually the most important factor for the quality of the sequencing reaction. In addition, the capillary sequencer runs best when you stick to a given concentration range. The best results are obtained with template concentrations of 1ng - 50ng for PCR fragments (depends on the size of the fragment) and up to a maximum of 300ng for plasmids (half these amounts for 10µl reactions). Please do not use more as this yields poor results and can block the capillaries!

Users who deliver too much DNA will be double charged!

[General code]

This code is designed to allow unequivocal allocation of sequencing results. It is based on an eight digit code to allow the easy conversion of file names between PC and Mac users.

First letter: affiliation (E = Entwicklungsbiologie; W = Genetik Weyertal; Z = Genetik Zülpicherstrasse; A = Andere).

Second and third letter: initials of the user (check for possibilities of overlap with other users).

up to five numbers or letters, no other characters

Example: WDT17857 or ZTLy2h22

[8-tube-strips / 20 microliter]

All reactions must be delivered in 0.2ml 8-tube-strips with strip caps, even if you have fewer than eight reactions (just leave the other tubes empty). This is essential for an efficient transfer of the reactions to the microtiter plates. If you do 10 microliter reactions, add 10 microliter water before delivery: the endvolume of 20 microliter is required for the standardized spin column purification procedure that is done at the facility. Samples can be frozen for over-night storage.

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[Mac format]

ABI has now shifted to PCs as computer standard. Mac users will therefore have to convert their sequencer files which can be done as follows:

1. Download and save "ConvProg.sit" from www.elimbio.com/Forms/ConvProg.sit
2. Expand the file to a folder "ConvProg Folder" using StuffIt Expander
3. Convert the PC electropherogram files from Windows format to Mac format using the conversion program "Sample File Win to Mac" in the ConvProg Folder:
   • Double click to launch the conversion program "Sample File Win to Mac".
   • Select the folder (NOT THE INDIVIDUAL FILES INSIDE THE FOLDER) generated from the above step 3), which contains the expanded sequencing results to be converted.
   • Click "Choose". The files will be converted in the folder automatically.

Note that the file names will only be properly converted when they follow the general 8-digit code.

[Network Access]

The results can be downloaded via ftp. Many browsers (e.g. Internet Explorer) support ftp options, but you may also use dedicated programs (for the Mac: Fetch 3.03 - http://fetchsoftworks.com/).

Use the following server address:

• entry2ccg.genetik.uni-koeln.de
• Username: client
• Password: client

You should never access the top level of this server. We can not hide the files on the top level, because the sequencer communicates itself via ftp. Although you will not be able to modify any files you must stick to the "Sequences" path, as indicated above.

You will see many folders which are labelled as follows:
e.g.: M_Run__2003-02-24_1

"M" is an alphabetically continuous variable that indicates the plate
"-_1" after the date is a numerically continuous variable that indicates the blocks of 16

When you open the folder you will see the following file names:

e.g.: M_A03_usernumber

"M" is same as above
"A03" refers to position in the plate

Your usernumber allows you to recover your sequence. You may need to open several folders before you find your samples. If you have difficulties, contact Karin Otto (4605).

Note: all sequences will be visible to all users. There is no secrecy. If you require secrecy, please contact us for special arrangements.

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