Instructions for Sanger sequencing
First you have to ask user accounts from bio-alf -at- helsinki.fi
Sequencing order forms:
For Helsinki University network users
For clients outside Helsinki University network
Our lab is located in Biocenter 2, Viikinkaari 5D, Helsinki (PL56). You can send your samples by mail or leave them in the post box at the lobby. (Door is open 8-16.00.)
If we have your samples before 11 am. in our box/lab, they will be run during next night (excluding force majeure; a machine breakdown). You will get the results as soon as we have checked the quality of the data, normally within three working days.
Send your samples in an envelope marked with order number and your name.
Template requirements
Quality:
Template quality and quantity are the most important factors affecting sequence length and quality. In more than 90% of failed sequencing reactions the cause is insufficient DNA quality. Errors in DNA quantification are also a recurrent problem. Quantification errors will not usually make the reaction fail completely, but they will seriously affect read lengths. Properties such as GC-content, repeats such as poly(A)-tails and loop structures affect sequencing reactions, so any information you can provide will help us select suitable reaction protocol.
Plasmids under 10 kb:
We require 5 µl of plasmid DNA for a single reaction in concentration of 100ng/µl. If plasmid is bigger than 10 kb, please refer to the procedures in large templates.
PCR products:
We require 100 ng (min. concentration 20 ng/µl) of purified PCR product for a single reaction. Products should be >150 bp. Always tell us the length of the PCR product. We can also purify the PCR products. If you have unpurified products, include the picture of the agarose gel electrophoresis with loading amounts. Always check your PCR fragments on agarose gel. If you have any secondary bands - however faint - the fragment must be purified from gel.
Large templates (cosmids, BACs, PACs)
We require 500 ng – 1µg of template DNA for reaction.
Primers
We have several standard primers for the most common vectors and for bacterial 16S rDNA sequencing. These primers are available at no extra cost.
If you want to use your own primers, take good care that you design them suitable for sequencing. The primers will be delivered in separate tubes at concentration of 5µM. The amount per reaction needed is 1µl. When designing the primer, remember that the readable sequence will not start right after the primer and the sequence quality might be poor during the first 10-20 bases. Allow about 50 bases between your primer and the site of interest.
Output formats and software links
Output formats
The primary output of the sequencers is graphical data, also called trace. The primary output is automatically interpreted by a program that gives text data, in other words the sequence output in letters (ACGT and IUPAC ambiguity codes). The graphic data is the most informative data, while the text data is prone to errors of interpretation due to imperfect primary data.
Traces (chromatograms)
Trace file as .AB1-file,
Text file as seq1-file, manual proof-read .The rawdata has been read through and the correction made, if necessary. Notice There is areas which are not very reliable, right at the beginning (1-30 bases) and 700 each.
Software that can open trace-files:
Sequence Scanner
Staden package
Chromas
Finch
BioEdit
Plate format
You can also send us samples at 96-plate format. There are some things to consider before making an order at plate format.
Primer
The primer has to be same in all samples.
Sample size
The size of the templates should be approx. the same. Don’t mix the PCR-product of 200 bp and plasmids at the same plate.
Concentration
We use the same volume of the templates for reaction. So make sure the concentrations are almost the same.
Sample order at the plate
Notice that we have 16 and 48-capillarymachines and the sample will be run at order starting well A01, B01, C01.., so fill in your plate from up to down, left to right.
Sample names
The samples will be named by well numbers, but it’s possible to add your own sample name to the file. We need a sample list at excel-form. You can send it by mail.
Data / results
The result files are big, therefore they can’t be sent as attachments. You can get your results on CD, you can come to pick up the data with memorystick or have a user account for a link to pick up the results for yourself.