SEQLAB The best thing that can happen to a DNA.

Transcription

1 SEQLAB The best thing that can happen to a DNA.

2 CONTENTS SEQLAB Who we are What we do GENOMICS Our sequencing service Your order (info and tips) Your data Your advantages Our purifying systems Our favourite thermocycler PROTEOMICS Our polyclonal antibodies Our protein identification and sequencing Our peptide synthesis SERVICE Price list Order forms

3 SEQLAB 03 WHO WE ARE SEQLAB is the DNA service in Germany. Our speciality is DNA sequencing performed by our highly effective service as seen in our tenders. SEQLAB, founded in 1996 possesses extensive experience in this field. No DNA is too sparse, no PCR-product too contaminated with secondary bands and no band shadow too difficult for us to clone. Five scientists and ten laboratory assistants are dedicated to fulfilling the prime purpose of this firm which is the delivery of highest quality DNA analysis of the most modern standard to all concerned in industry, university, clinic and organizations for private research. 6 Milagros Lazo Silva 2 Dr. Andreas Wolf Martina Dreier 7 Dr. Bernd-Peter Ernst 12 Dr. Michael Betzler 13 Jasna Fraatz Secondary structures, inverted repeats, homo- and heteropolymeric stretches, high GC or ATs We are not saying they are no problem but we do say that, as sure as the Gänseliesel of Göttingen is the most kissed girl in Germany, we can solve them. If you give us a true sample of DNA for our Full Service we will not rest until the sequence lies open. Promise! Dr. Werner Baussmerth 4 Dr. Mustafa Benli 5 Dr. Otto Knobloch Katharina Dörnte 9 Dr. Rüdiger Kind 10 Dr. Karla Busch 11 Dr. Poulad Monzavi

4 04 SEQLAB WHAT WE DO We are DNA service providers and have set as kernel of our competence the sequencing on commission in our differing, individually relevant proffered services: The HotShot is the ace in SEQLABs history. Although fluorescent colour band sequencing has been available since the 90s and was offered by various sequencing centres the costs remained so high that many smaller laboratories and university departments preferred to use their own resources. This involved radioactive methods as a rule, and the yield, following two days of heavy work, was information on a modest 250 bases. The breakthrough occurred in 1999 with SEQLABs HotShot primer and the DNA for sequencing were sent ready mixed and the savings in work time were passed on to the client in the form of reduced costs. Thus sequencing within one s owns four walls became even less attractive economically. Since it was not possible to repeat the cycle sequencing a suitable cycle sequencing (CS) protocol had to be developed to enable the sequencing of DNAs including those with simple secondary structures to be carried out successfully at the first try. This protocol has been available since 2000 and constantly delivers good results. 97 % of clean DNAs with no ex-cessive secondary structures deliver 99 % exact readings. AdvantageRead is HotShot s big brother and fills the gap towards the FullService. Based on the same CS protocol it renders none the less possible a repeat evaluation under optimal conditions through a separate delivery of primer and DNA substrate should the first attempt be inconclusive and deliver poor results, due to unsuitable secondary structures. The FullService is a more advanced step. Our experience at SEQLAB in chemistry and the CS evaluation enables us to identify just about every sequence, thus bringing us to the reputation of problem solvers. There are virtually no DNAs with secondary structures which find their way to SEQLAB and remain undeciphered. Apart from the sequencing described we also offer a wide spectrum of further services and products: GENOMICS Nutcracker sequencing of hairpin DNA structures; direct sequencing of cosmids, fosmids, BACs and PACs, whole genome sequencing, shotgun sequencing of larger constructs; primer design and synthesis, in vitro amplification, plasmid preparations; DNA purification systems for many purposes; PROTEOMICS Protein sequencing and identification; Polyclonal antibodies from various animal sources; peptides with and without modifications.

5 GENOMICS 05 OUR SEQUENCING SERVICES Customer s work SEQLAB s work ca. 300 b HotShot 9C 6 g 7.95 Eg^b Z ca. 900 b + BIGDYE %#' ba " IjWZ ca. 650 b C 6 AdvantageRead 24-h-Service + PRIMER + BIGDYE ca. 900 b Posted usually within 24 h (+repetition if necessary) C 6 FullService* S ca. 350 b A ca. 650 b + PRIMER + BIGDYE B ca. 900 b * BACS, PACS AND GENOMIC PROJECTS ARE HANDLED AS FULLSERVICE. Posted usually within 24 h incl. basic editing (+repetition if necessary)

6 06 GENOMICS YOUR ORDER (INFO AND TIPS) The delivery of the sequence data follows usually 24 hours after receipt of the post (24 hour service). The samples usually arrive before pm, are put into the cycle sequence reaction the same day and into the sequencer that evening. Results are read and posted the following day. HotShot sequencing For the HotShot sequencing you send us the template and the primer of choice (already mixed) in a PCR tube of 200 µl with a flat lid. The PCR cups should be numbered on the lid beginning with 1 and then sequentially. Total volume should not exceed 7 µl: DNA in 10 mm TRIS, ph 8.5; 20 pmol primer. HotShot samples should not be wrapped in parafilm or foil. Necessary DNA quantity FOR HOTSHOT SEQUENCING For plasmid DNAs please send µg. For PCR products the following quantities should be sent: length of fragment divided by 4 = quantity in ng, e. g.: PCR product ~500 bp : 4 = ~125 ng; a little extra is no disadvantage. The primer should be one of 18 25mer with a Tm of C. Please observe these exact instructions for the HotShot sequencing. They are indispensable for the successful automatic work programme. FOR ADVANTAGEREAD SEQUENCING Plasmid DNA: µg with a concentration of ng/ µl. PCR products: quantity dependent on the length with a concentration of: PCR fragment length divided by 20 = concentration ng/µl; e. g.: PCR-Product ~500 bp : 20 = ~25 ng/µl. FOR FULLSERVICE SEQUENCING ds-dna (bp) optimal amount of DNA (ng) required concentration of DNA (ng/µl*) , , , , , ,5 FOR PCR PRODUCTS As a rule of thumb: for DNA quantity: PCRfragment length divided by 4 = DNA quantity in ng; for the concentration: PCR length divided by 20 = concentration in ng/µl. For the sequencing we need 3 to 4 times the amount of the calculated DNA quantity, so that the possibility of repeating the sequence reactions is given, should need arise. FOR PLASMIDS 3 8 µg DNA depending on the size of the vector. Minimum concentration is 200 ng/µl; for larger plasmids (vector + insertion > 8000 bp) is a concentration of ng/µl recommended. Please dissolve the DNA in Tris/Cl, 10mM, ph 8.5!

7 GENOMICS % GC - content 82.3 % GC - content Precipitation of DNA Please precipitate the DNA solely at room temperature; the reagents employed should also be stored at room temperature. Salts tend to precipitate at lower temperatures, thereby disturbing the sequencing. The DNA should in addition be well washed with at least the same volume of 70 % ethyl alcohol, with which the precipitation is undertaken. Please leave the DNA precipitate for 5 10 minutes fully under 70 % ethanol; preferable is a second washing. Plasmid preparations For a specimen of plasmid DNA to deliver excellent sequence results the following points should be observed: 1) culture medium residues carried over in the preparation lead to bad results. The medium must be completely removed following the centrifuging down of the cells. We recommend therefore the washing of the cells in Tris/Cl-puffer solution (10mM, ph 8.5) and then the complete removal of the puffer. 2) Overloaded Mini-, Midi- and Maxi-Kit columns deliver comparatively large amounts of DNA, the sequencing of which is, however, difficult or impossible. We recommend therefore the purification by column of minipreps max. 2 ml, of midipreps max. 20 ml and of maxipreps at the most 100 ml cell suspension. As an alternative for low copy plasmids: up to 10 ml cell suspension for a miniprep, washing of the cells with Tris/Cl buffer (10 ml, ph 8.5), and then a second miniprep with µg plasmid DNA from the first preparation. PCR products The following tests improve the probability of obtaining good sequence readings from PCR products: a) It should be possible to reamplify a good yield of the PCR products; should the PCR product band only be weakly visible by the reamplification then the sequencing will itself be poor. b) A product should result only with both PCR primers; to test this you can lay down two PCR reactions and use the forward primer in one and the reverse primer in the second. Should a product result by one of these tests, then the corresponding primer was used as the forward and also as the reverse primer (so called one-primer PCR ). We note this problem frequently with PCR products of genomic DNA. c) PCR products should be cleared of superfluous primers and dntps as well as of any primer dimer products to avoid influencing the following sequence reactions. OVER 90 AVAILABLE PRIMERS are listed on our internet page under services, link: available primers. You can speedily find there which primers are appropriate for your vector. The use of these primers is included in our offer on your commission of Advantage-Read or Full Service. Primerdesign/ -synthesis Primers can also be ordered. Simple and advantageous primers (up to 25mer) are offered in conjunction to a sequencing commission. PACKAGING OF THE SAMPLES FOR TRANSPORT BY POST: Please use padded envelopes for your post. Since these are not a 100 % protection against breakage the tubes should be additionally packed in plastic boxes, falcon casings or small cardboard boxes. HotShot samples should be solely sent in 200 µl cups with a flat lid. These must not be wrapped in parafilm or similar foil! The DNA samples for Advantage-Read and FullService sequencing commissions should be sent in 1.5 ml cups (the lid alone wrapped with parafilm) or in 1.5 ml screw cap vessels.

8 08 GENOMICS YOUR DATA 1. Online service SAFE, FAST, TRUSTWORTHY Commission and delivery of sequence data is usually by means of our server system SEQLABdirect. To use SEQLABdirect you only need your customer s number and a password. We inform you of your customer s number following your first written order. The password is sent automatically via at the next commission. In case of large amounts of data we will compress the sequence files, in order to fit more information into an . The compressed files have the ending *.sea. On Mac-Computers they are decompressed automatically by a simple mouse click. On PCs you will have to decompress the files with stuffit. 2. Other delivery possibilities Programmes for data editing The following simple programmes for observance and editing are on the internet: for PCs (freeware): CHROMAS Lite 2.01 from Technelysium: chromas_lite.html for MACS (freeware): EDIT VIEW from Applied Biosystems: support/software/ For decompressing files: for PCs (freeware): STUFFIT from SmithMirco Software The data can be sent per should you so request. The four colour prints of the electropherograms on paper or the delivery of the data on a CD-ROM or a diskette is possible for a slight extra charge (see current pricelist). SEQLABS ONLINE SEQUENCES You can also call up your sequence results from any internet computer without recourse to special software (Chromas, EditView etc.). An exclusive SEQLAB service. YOUR ADVANTAGE Prepaid HotShots On subscription of Prepaid HotShots we give up to 36 % discount. Please ring us on to arrange your prepaid account. Following the establishment of your prepaid account you can only commission Prepaid HotShots online. Written orders are unfortunately not possible. All other HotShot instructions are unaltered Dr. Werner Baussmerth

9 GENOMICS 09 OUR PURIFYING SYSTEMS In the course of development of the DNA preparation methods the original phenol/chloroform DNA extraction process according to Maniatis was soon superceded by procedures which function without phenol and which are based Miniprep-Plasmid-Kit on silicon- and other types of The Miniprep-Plasmid-Kit delivers more quickly than a conventional Plasmid-Kit a solid amount of highly pure DNA. membrane allowing a selective binding of DNA. These procedures are justly regarded as having the highest standard on the present day market and as being mature. Notwithstanding this we have succeeded in raising the technical standard yet again: a strict procedure entailing a much shorter time of preparation delivers a constantly high DNA yield of superb quality. Clean DNA: excellent sequencing quality with single and double repeat-motives SLH-Low-CopyPlasmid-Kit This Kit is used in the extraction of plasmid DNA from low copy strains. 10 preparations, article no.: preparations, article no.: preparations, article no.: The SLH-Low-Copy-Plasmid-Kit delivers in low copy plasmids a solid amount of highly purified DNA and combines the high yield of classic plasmid preparation methods with the high purity and extraordinary quality of a process based on the affinity method. 10 preparations, article no.:

10 10 GENOMICS PCR-PURIFYING-KITS PCR-Purifying-Kit (2-STEP-PROCESS) You use our PCR-Purifying-Kit for a fast purification and concentration of PCR reactions in only two stages. Adding the binding buffer to PCR solution 1. Step: Binding of PCR fragments 2. Step: Elution of PCR fragments Employ the advantages of the PCR-Purifying-Kit to enjoy the simple and fast working method as well as the excellent quality of the purified DNA. With the PCR-Purifying-Kit it takes less than five minutes to obtain a quick and easy purification. The 2-step-process (binding and elution) is exceptionally suitable for fragments of between 80bp and 30kb. The kit has flexible elution volumes, down to a minimum of 10 µl at its disposal and is also suitable for the concentration of samples. The Gel-Extraction-Kit is used for a rapid extraction of DNA fragments out of TAEand TBE-gels. In approximately one quarter of an hour it achieves close to 100 % extraction and the highest quality and purification of the fragment The Gel-Extraction-Kit is the method of choice where the pureness and unicarity of the DNA is at stake. 10 purifications; article no.: extractions, article no.: purifications, article no.: extractions, article no.: purifications, article no.: extractions, article no.: Gel-Extraction-Kit Those PCR products which appear in the gel as a band may also be accompanied by primerdimers (and other secondary products) in just non-visible concentrations. These can interfere, due to thermodynamic promotion, either with the cloning of the PCR products, or cause double sequencing of the product in the first bases, due to cosequencing of the dimer. PCR-Combi-Kit (PCR PRODUCT PURIFICATION OUT OF SOLUTIONS OR GELS) The PCR-Combi-Kit makes a highly efficient purification of DNAs out of reaction solution and agarose gels possible. In both operations the PCR-Combi-Kit delivers highly pure DNA of exceptional quality 10 purifications, article no.: purifications, article no.: purifications, article no.:

11 GENOMICS 11 Plant-DNA-Kit RNA-Mini-Kit Bacteria-DNA-Kit Our Plant-DNA-Kit enables up to 100 mg of genomic DNA to be isolated from varying vegetable material. Following the lysis of the vegetable substance with consecutive purification from unsoluble components and unwelcome contaminants (e. g. phenols) binding of the genomic DNA by means of a filter cartridge and the elution of highly pure DNA is achieved. Our RNA-Mini-Kit enables the speedy and efficient isolation of the total RNA out of eukaryotic single celled organisms, bacteria and macerated tissue. Following the lysis of the original substance a DNase I free selective purification of the genomic DNA is carried out. The RNA is then repeatedly washed and eluted. The obtained total RNA is of excellent quality and outstandingly suitable for all further applications. Our Bacteria-DNA-Kit enables the speedy and efficient isolation of bacterial genomic DNA out of pellets of grampositive and negative bacteria from young cultures. Following the initial lysis step and the consequent proteinase K digestion of the proteigenic cell residues the liberated DNA is bound to the membrane of the affinity filter cartridge. The bound DNA is then eluted with a good yield and in outstanding quality. 10 preparations, article no.: preparations, article nor.: preparations, article no.: preparations, article no.: preparations, article no.: preparations, article no.: preparations, article no.: preparations, article no.: preparations, article no.: Dr. Otto Knobloch according to ABI user protocol Do you find this a hard nut to crack? by SEQLAB... just leave it to us!

12 12 GENOMICS OUR FAVOURITE THERMOCYCLER At SEQLAB we have worked with many thermocyclers. From the first time on that we tested the SENSOQUEST LabCycler we were positive: this one and no other for us. Its precision, quality and flexibility speak for themselves since it combines in one all those advantages that one has always looked for in a thermocycler. In addition it also looks good. Arrange for us to set one up for trial in your rooms and see if it doesn t convince you: this also is your thermocycler! THERMOCYCLER Temperature: -5 C to C Heating rate: 4.2 C per second Cooling rate: 3.2 C per second Optional with gradient: 20 C (+/- 10 C) Autoresume function Heating lid: automatic, temperature and pressure are programmable Electrical potential: 85 V to 265 V without switching over Length: 444 mm, width: 251 mm, height: 201 mm LabCycler Basic (without gradient): LabCycler Standard (with gradient): Dr. Bernd-Peter Ernst USERS SURFACE Languages: German, English Protocol function Intuitive service software TFT touchscreen Coloured graphic display RS232 interface for software updates Gradient upgrade: THERMOBLOCKS Electro formed gilded silver Processual steered gradient capable block with individually steered peltier elements for a particularly homogenic temperature with high heating and cooling rates. Unique universal alternation block system 48-wells block for individual tubes (0.5 ml) 96-wells block for microtitreplates 96 or individual tubes (0.2 ml) 384-wells block for 384 microtitreplates Thermoblock 48: Thermoblock 96: Thermoblock 384:

13 PROTEOMICS 13 OUR POLYCLONAL ANTIBODIES For the antibody production we work with rabbits, mice, rats and guinea pigs. If you wish you may also choose chicken, goat or hamster. Place your order with SEQLAB informally, a special order form is not necessary. The following sample protocol can be varied to suit your wishes: before the immunisation has begun you will receive 2 to 5 pre immune sera which you can test for possible cross reactions. You receive 2 ml testaliquot after each blood withdrawal. Standard immunisation scheme (3 months) Standard immunisation scheme (2 months) Day 0 1. Injection (preimmune serum >5 ml) Injection Day 0 1. Injection (preimmune serum) Injection Blood withdrawal (10 20 ml) Blood withdrawal (10 20 ml) Injection Injection Blood withdrawal (2 ml a ml) Blood withdrawal (2 ml a ml) Injection 60 Exsanguination 98 Exsanguination ALSO ON OFFER: Antibody labeling (HRPO, biotin, fluorescin) Quality check under enzymatic THE NECESSARY ANTIGEN FOR ANTIBODY PRODUCTION IN RABBITS: Form of antigen 1. Injection Follow-up injection Protein > 20 kd Protein < 20 kd µg µg 100 µg µg Insoluble protein, microbodies polysaccharides µg µg µg µg Peptides linked to carrier molecules (BSA, OVA, KLH) µg µg Non viable bacteria, viruses, yeast protein Please deliver antigens in physiological puffer solution (e. g. PBS, ph 7 8), lyophylised, in a PAA sample or blotted onto nitrocellulose membranes. Volume of antigen required: µl per injection. control and HPLC/SDS-PAGE or ELISA analysis Antibody purification: affinitypurification by means of protein A- or protein G-sepharose Affinity purification of sera by means of an antigen matrix Antibody fragmentation, FAB fragments ELISA test in a 96-wells antigen microtitreplate Dr. Werner Baussmerth

14 14 PROTEOMICS OUR PROTEIN IDENTIFICATION AND SEQUENCING SEQLAB offers a mass-spectrometric (MALDI-TOF) analysis for the measurement of the purity and molar mass of proteins, as well as for protein sequencing by the degradation method of Edman. Protein identification In the mass spectrometric analysis for the measurement of purity and the molar mass of proteins you can choose between a total mass measurement and a fingerprint following trypsine digestion. Simply send us a cut out SDS band in a cup. To prevent desiccation add 2 drops of water with a pipette. Protein sequencing after Edman For the protein sequencing according to Edman the individual amino acids are split off and determined one after the other. OUR SERVICE COMPRISES: Chromatogram and process data of the separate runs Log data for the individual amino acids Declaration of the determined amino acid sequences Nomenclature and charge numbers of the reagents employed, should this be desired REQUIREMENTS FOR THE SAMPLES TO BE SEQUENCED: The samples should be lyophylised If possible, please send the relevant chromatogram A definite coomassie band should be visible in blot samples Sample quantity ca. 1 µg, or, for more extensive commissions, up to 5 µg. Use solely PVDV membranes for blotting The sample should have as little as possible contact with acetic acid, since otherwise sequencing may not be feasible. HPLC-separation of PTH-amino acids Delivery time: around 2 3 weeks Dr. Rüdiger Kind

15 PROTEOMICS 15 OUR PEPTIDE SYNTHESIS Peptide syntheses are also offered by SEQLAB. Synthesis is carried out by the solid-phase-method under the employment of the most modern synthesis machines. We proceed according to the FMOC method. Peptide synthesis You may choose between different purity levels: Above 70 % peptide purity is sufficient for the production of polyclonal antibodies ( immunisation ) by various animals. Purity stages above 80 %, over 90 % and over 95 % are necessary for more demanding immune studies in vitro and in vivo. All peptides are purified with HPLC. A mass spectrogram is delivered with each peptide, thus enabling an exact identification to be made. In addition we offer specific modifications of the peptides such as MAPS, MAP-4, D-aminoacids, N-acetylisation and C-amidisation. Phosphorylisation with phosphoserine and phosphothreonine can also be carried out. Further modifications are available on request. Conjugations on various carrier proteins can also be performed. As a rule the conjugation of ca. 2 3 mg peptide and BSA or KLH is undertaken. Please enquire as to further carriers. We check if the sequence that you request involves changes in the standard synthesis method on receipt of your commission. Should these cause an increase in the cost you will, of course, be informed immediately. Delivery time Peptide syntheses with HPLC purification up to 20 AS are carried out within 4 weeks. Longer peptides and such with modifications or difficult amino acids can take up to 6 weeks Dr. Werner Baussmerth

16 16 GENOMICS PRICE LIST VALID FROM 20TH FEB. 2007, PLUS VAT Order no Service offer Description and comments Single tube HotShot sequencing HotShot (read length ca. 300 b) Extended HotShot (read length ca. 900 b) Prepaid HotShot sequencing 50 HotShot < 300 bases 100 HotShot < 300 bases 500 HotShot < 300 bases 1000 HotShot < 300 bases 50 Extended HotShot < 900 bases 100 Extended HotShot < 900 bases 500 Extended HotShot < 900 bases 1000 Extended HotShot < 900 bases HotShot sequencing in microtitreplates 384 well HotShot ca. 300 bases 96 well HotShot ca. 300 bases 384 well HotShot ca. 900 bases 96 well HotShot ca. 900 bases ADVANTAGE READ Advantage 650 (ca. 650 bases) Advantage 900 (ca. 900 bases) PREPAID ADVANTAGE 650/ Advantage 650 (length ca. 650 bases) 100 Advantage 650 (length ca. 650 bases) 500 Advantage 650 (length ca. 650 bases) 1000 Advantage 650 (length ca. 650 bases) 50 Advantage 900 (length ca. 900 bases) 100 Advantage 900 (length ca. 900 bases) 500 Advantage 900 (length ca. 900 bases) 1000 Advantage 900 (length ca. 900 bases) FULLSERVICE SEQUENCING Short run S (ca. 350 bases) Standard run A (ca. 650 bases) Long run B (ca. 900 bases) We can only guarantee these very low prices, if you exactly fulfil all the conditions: put the appropriate amount of DNA dissolved in 5 10 mm TRIS- Cl, ph plus 20 pmoles of the primer in a total volume of 7 µl together in a 200 µl PCR tube with a flat lid. Label the tubes only on the lid with consecutive numbers and don t wrap them with parafilm etc., but put them in a stable box or plastic tube for protection and place this assembly in a shock-absorbing padded envelope. We need 0.6 µg of plasmidary DNA and 25 ng for each 100 bases of length of PCR-products (e.g. 50 ng of a 200 bases PCR-product). The resulting sequences are not edited, the reactions can t be repeated. An invoice will be issued irrespective of the sequencing result. Resulting sequencing data and electropherograms at SEQLAB Server. First order on the HotShot order form only, then please use our online service at Hot Shots in microtitreplates (MTPs) should be prepared same as above; customary MTPs schuld be filled with components and covered with an adhesive film. Label the plate on the margin of the MTP and on the film with a summarized name. The resulting sequences are not edited, the reactions can t be repeated. Cycle sequencing, universal or specific primers, capillary electrophoresis on the 3730 ABI automatic sequencer; resulting sequencing data and electropherograms at SEQLAB Server. The AdvantageRead closes a gap between the HotShot and our FullService Sequencing, the latter of which offers 3 different read lengths (No S to 3000 B). Although it includes an extended treatment compared to the HotShot (i. e. repeated cycle sequencing with different primer and/or DNA amounts), it falls behind the sophisticated trouble shooting of our FullService Sequencing procedure. First order on the Advantage order form only, then please use our online service at Cycle sequencing with universal primers, gel elelectrophoresis, basic verification, sequence data and electropherogram via . The resulting sequence is preliminarily edited. All Ns in the start of peaks are removed from the sequence and the whole sequence is cleaned up from gross irregularities by individual editing. The sequence data will be delivered to you by with a short comment. All sequencing data are kept in the database for 3 months at SEQLAB, allowing you the opportunity to order further walking beyond those delivered sequences. Primer walking; cycle sequencing, electrophoresis, sequence data and electropherogram via . The resulting sequences are fully edited, additional alignment at 99,99 % fidelity. The expected sequencing error rate is 0.1 % per base for single strand primer walking and < 0.04 % per base for double strand primer walking. Cycle sequencing with universal primers, gel electrophoresis, basic verification, sequence data and electropherogram via . SEQLAB has the rich know-how for sequencing large constructs and can perform sophisticated sequencing protocols, leading to highly successful sequencing of most BACs, PACs and genomes. Unfortunately there is no warranty for success despite this optimisation. By genomic direct sequencing is not possible to tell wich length were available. Price Ask for prices 6.20/well 6.90/well 9.10/well 9.90/well Ask for prices 25.50* 30.70* 35.90* * Incl. OD- and GC-extra charge PRIMER WALKING Single stranded sequencing Sequencing of both strands in publication quality > 1000 bp BACS, PACS direct sequencing BAC-S (ca. 350 bases) BAC-A (ca. 650 bases) BAC-B (up to 900 bases Direct sequencing of genomic DNA Genomic walking (in situ positioning) Primer design Primer synthesis Projects, genomic analyses Shotgun sequencing, large inserts Plasmid preparation in MTPs 96 miniprep in microtitreplates Plasmid preparation Miniprep Column purification Using membranes for purification of plasmids, PCR products, Cosmids, BACs or PACs Purification of PCR products (Ag.) Agarose gel purification or spin column Purification of PCR products (PAA) High resolution gel separation and extraction. It is possible to discriminate DNA fragments with only 2 bp length difference on polyacrylamide (PAA) gels p.b p.bp For the determination of the insertion locus of a known DNA section in its genomic surrounding, Ask for prices or also for sequencing from a known DNA section into the unknown At primer selection we receive its highest biological specificity by using modern primer design programs with subsequent individiual critical examination of the proposed sequences. Our long years of experience help us choose the right ones. Primer, 10 nmol scale, up to 25mer primers from 8.00 on Price lists also available as PDF at Ask for prices 2.00/well

17 GENOMICS PRICE LIST VALID FROM 20TH FEB. 2007, PLUS VAT Order no Service offer Description and comments Purification of PCR products in MTPs 96 well purification of PCR products in microtitreplates In vitro DNA-amplification In vitro DNA-amplification Quality control of DNA OD (concentration and purity); agarose gel or polyacrylamide gel (per fragment) DNA precipitation With ethanol Cup-to-cup transfer Transformation from unsuitable cups to suitable ones Concentrating of DNA Concentrating of samples with < 200 ng/µl Sequence verification (full editing) Alignment Nutcracker Firstly, this feature consists of the trimming of the raw data for the first uninterpretable 10.00/file oversteered signals at the beginning of the sequence and those uselessly low signals at the end of the sequence. Both kinds of signals disturb the computational integration of the desired peaks. Then, we remove all of the Ns at peak start, which are derived. From system immanent electrophoresis failures. Subsequently comes the careful examination of 10.00/file the whole sequence including the manual correction of base calling failures and identifying of real (biological) irregularities. Attempt with special arrangement from 250. on Plasmid preparation kit SLH low copy plasmid kit 1.00/well (*) is incl. in Full Service Delivery and packaging Delivery and packaging Mail (abroad, without Antibody delivery) Set-up charge Set-up charge direct genomic sequencing These charges will be invoiced only if the repeated sequencing attempts have failed even under specialised conditions because your DNA was dirty or degraded or the primer was wrong. Only if the direct genomic sequencing has failed totally. Gelextraction kit PCR combi kit Plant DNA kit RNA mini kit Bacteria DNA kit HotShot cups GC-special Data transfer * 3.50 Kit for 10 minipreps via a new membrane for faster preparation of highly pure plasmids (P) Kit for 50 minipreps Kit for 250 minipreps Kit for 10 low copy Plasmid preparations via a new membrane (P) Kit for 10 PCR purifications with a new affinity based membrane in two steps (P) Kit for 50 PCR purifications Kit for 250 PCR purifications Kit for 10 extractions via a new affinity based membrane in tree steps for faster preparation of highly pure DNA Kit for 50 PCR purifications Kit for 250 PCR purifications Kit for 10 DNA purifications from solution or gel in two or tree steps Kit for 50 DNA purifications Kit for 250 DNA purifications Kit for 10 DNA preparations from plant cell for faster preparation of highly pure DNA Kit for 50 preps Kit for 250 preps Kit for 10 RNA preparations with a new affinity based membrane Kit for 50 RNA preparations kit for 250 RNA preparations Kit for 10 bacteria DNA präparations with a new affinity based membrane Kit for 50 bacteria purifications Kit for 250 bacteria purifications 500 PCR cups with flat lid for HotShots 1000 cups GC-special for inverted repeats, GC extra rich sequences, sequences with particular tricky secondary structures. Attempt with special arrangement. Mail (domestic, without Antibody delivery) PCR purification kit Price Data delivery normaly at the SEQLABdirect server Via diskette Via CD-ROM Via Via four colour print incl. in Full Service 5.00/ free /page Price lists also available as PDF at

18 18 PROTEOMICS PRICE LIST VALID FROM 20TH FEB. 2007, PLUS VAT Order no Service offer Description and comments Price Immunization of rabbit, guinea-pig, Polyclonal antibodies, raised in rabbit, guinea-pig, hamster; chicken, hamster 2 months protocol with your peptide/protein as antigen. as noted above in 3 months protocol Immunization of mouse Polyclonal antibodies, raised in mice; 2 months protocol /animal Immunization of rat Polyclonal antibodies, raised in rat; 2 months protocol /animal Immunization of goat Immunization incl. peptide synthesis and coupling Polyclonal antibodies, raised in goat; 2 months protocol. each additional month 125,00 EUR Immunization of 2 animals with artificial peptides as antigen including peptid synthesis and coupling; 3 months protocol /animal Preimmune serum Per animal and week 10.00/animal Deliveries for immunization For all deliveries of an immunization project Peptide synthesis 70/10 10 mg, over 70 % pure, all peptides will be purified using HPLC /aa Peptide synthesis 70/20 20 mg, over 70 % pure, HPLC 29.80/aa Peptide synthesis 80/10 10 mg, over 80 % pure, HPLC 28.00/aa /animal /animal Peptide synthesis 80/20 20 mg, over 80 % pure, HPLC 45.90/aa Peptide synthesis 90/10 10 mg, over 90 % pure, HPLC 39.90/aa Peptide synthesis 90/20 20 mg, over 90 % pure, HPLC 58.80/aa Peptide synthesis 95/10 10 mg, over 95 % pure, HPLC 48.80/aa Peptide synthesis 95/20 20 mg, over 95 % pure, HPLC 69.70/aa Modification of peptides (B) Modification of peptides (N10) Modification of peptides (N20) Modification of peptides (C10) Modification of peptides (C20) Phosphorylation of the peptide with phosphotyrosine, phosphoserine or phosphotheronine Biotinylation of peptides N-acetylization 10 mg 20 mg C-amidation 10 mg 20 mg Phosphorylation 10 mg 20 mg Other modifications of peptides Labeled peptide Conjugation Coupling of the peptide with BSA or KLH, conjugation will be made with 2 3 mg peptide Protein sequencing up to 4 AA N-terminal, Edman s degradation per amino acid Protein sequencing > 4 AA Protein identification (MT) N-terminal Proteins are enzymatically cleaved; cleavage products are then MALDI-TOF analysed. Only proteins the charactarizations of which are already deposited in databanks can be identified /aa Setup cost protein sequencing In case of missing results of protein sequencing Setup cost MALDI-TOF In case of missing results of MALDI-TOF Price lists also available as PDF at Ask for prices 80.00

19 Country Department Order number or date diskette CD-ROM signature for MAC *.sea for PC *.zip DATA TRANSFER Preferably online delivery of data from our server! If desired otherwise, please tick the following forms of delivery: (compressed if necessary) Sequence Laboratories Göttingen GmbH Hannah-Vogt-Straße 1 D Göttingen Postfach 3343, Göttingen Phone: +49 (0) Fax: +49 (0) Bio-Service@seqlab.de primers should be 18 to 20mers with a Tm from C. Concerning different templates use the following amounts of DNA: plasmids: µg PCR-product ~1000 bp: 250 ng PCR-product ~500 bp: 125 ng PCR-product ~200 bp: 50 ng Please mix the necessary amount (s.b.) of DNA in 5 10 mm TRIS/Cl, ph 8.5, and 20 pmol of primer in a total volume of 7 µl. remarks SEQLAB offers the highest quality (with a perfect DNA up to 99.9 % reading reliability) and guarantees absolute privacy of the data. number tubes on the lid successively with two-place figures (01 96) Extended HotShot (app. 900 b) * *) no mixed orders please: use separate order forms Standard HotShot (app. 300 b) * AMOUNT OF DESIRED HOTSHOTS For HotShots please send us: A 200 µl PCR tube with a flat lid, containing the DNA and the primer for sequencing. (already mixed) 2 1 data ATTENTION: We can only guarantee these very low prices if you exactly fulfil all the conditions mentioned above. In case of a formal mistake, you can appoint the samples as AdvantageReads, but we can`t send them back. If the amount or quality of DNA or the primer are suboptimal, this may lead to a total failure of the sequencing reaction. However, payment for HotShot sequencing is obligate irrespective of the result. If the category is not ticked, the order will be regarded as Extended HotShot order. Don t wrap any parafilm etc. around the tube, but put it into a stable box or plastic tube as protector and place this assembly in a wadded envelope. Fill in this order form carefully and pay attention to primer and template concentrations and amounts! Dear customer, please use this order form only for the first order, after that please use our online service at Fax City Street Phone Company/Institute Name ORDER FORM HOTSHOTS

20 Department Company/Institute ORDER FORM ADVANTAGEREAD Name Country City Fax Street Phone Order number Dear Customer, please fill in this order carefully and pay attention to primer and template concentrations, use the same name on the tubes as in the order form! After purification please dissolve the DNA in 5 10 mm TRIS, ph8.5 (no EDTA)! Wrap the tube tops with parafilm and send the samples well protected per mail. You need not send universal primers, but send specific primers in an extra tube. length (kb) reading length remarks ATTENTION: The Advantage 650/900 holds a second attempt with optimised DNA/Primer-relation out. Please, order the analysis of more difficult templates on an extra order form as FullService. Those templates will be treated intensively with specific SEQLAB-protocols (s. price list, No and 80072). NEW are the different lengths. Please mind that an unticked order will be processed as Advantage 900. vector 900 b template data 650 b 900 b primer name PCR 650 b 900 b template name Pl PCR 650 b 900 b please tick 1 Pl PCR 650 b 900 b vector/insert PCR fragment 2 Pl PCR 650 b 900 b distributor cloning site 3 Pl PCR 650 b please write in block letters, max. 6, but no greek letters 4 Pl PCR kind, concentration, quantity we need per reaction: Pl asmid ( ng/µl), 10 µl PCR-Produkt (~bp-länge/20 = ng/µl), 10 µl 5 Pl Tm= C; 18 25mer; 10 pmol/µl, each 10 µl 6 More than 100 universal primers are available, e. g.: T3, T7 promotor, T7 terminator, M13 forward -21, M13 rev, SP6, U19, pgex 3, pgex 5, pgl 1, pgl 2 signature DATA TRANSFER Preferably online delivery of data from our server! If desired otherwise, please tick the following forms of delivery: (compressed if necessary) diskette CD-ROM for PC *.zip for MAC *.sea colour print fax date Sequence Laboratories Göttingen GmbH Hannah-Vogt-Straße 1 D Göttingen Postfach 3343, Göttingen Phone: +49 (0) Fax: +49 (0) Bio-Service@seqlab.de