Frequently asked questions (FAQ)


    FST Production

    6. Database Search

    15. Seed Ordering

  16. Why do we sometimes receive seeds very long after an order?

    28. Miscellanies


  1. What is the genetic background of GABI-Kat lines? Columbia-0
  2. Which vectors have been used to create which lines? We have used four vectors: pAC106 (GenBank:AJ537513), pAC161 (GenBank:AJ537514), pGABI1 (GenBank:AY529716) and pADIS1 (GenBank:AY529717). Sequence and overview map data of all vectors are available from the download page. Features of interest which are not included in the map should be deduced from the sequence. For a specified line, the vector is displayed in the "Show Sequence" page of SimpleSearch. The link between block number and vector is as the follows (block number is the first 3 digits of the line ID and '-' is inclusive):
    blocks 001-032, 801-849 => pAC106
    blocks 500, 566, 567, 579, 580, 587, 588, 597, 645, 654, 658-660, 662, 664, 666, 692, 693, 699, 700, 702-739, 741, 748, 763, 764, 774-776 => pGABI1
    block 568 => pADIS1
    all other blocks => pAC161
  3. Where can I get more information about vectors and primers used in GABI-Kat? You can get more information about vectors and primers in the vector and primer info page.
  4. What is the selection scheme? The answer is on the sul selection scheme page.
  5. How was the flanking sequences generated? Please see the sequencing strategy page. More details can be found in paper: Strizhov N et al. BioTechniques. 2003 35:1164-8. [PubMed]
  6. What do I need to know to use SimpleSearch? To use GABI-Kat SimpleSearch, you need to know the A. thaliana AGI gene code (e.g. At3g55120 for chalcone flavanone isomerase) for any AtYFG (Your Favourite Gene from A. thaliana). You can use TAIR or mips (MAtDB v2) to find these codes for any annotated AtYFG. There is also a sequence-based (BLAST) search option integrated into SimpleSearch; you can search with the DNA or protein sequence from your favourite gene.
    Note: see FAQ 12 for the genome annotation version we are using.
  7. How can I evaluate the sequence which defines the hit? First, you need to check which primer was used to generate the respective sequence. This information is encoded in the designation of the sequence: the last field contains the primer name (e.g. 8409 in 65-K012471-022-054-A09-8409). Many, but not all, sequences were generated with primer 8409 which reads out of the left border (LB). A list of all primers and the respective sequences is available from the GABI-Kat sequencing strategy page. Second, use the FST sequence to confirm our automatic annotation of the FST. From the FST sequence and the information on the (potential) detection of T-DNA sequences at the beginning the FST read in the "T-DNA Border" field, you can deduce the approximate insertion location. Please note that the insertion position deduced from FSTs is not absolutely reliable, especially when no T-DNA sequence is detected at the beginning the FST (and also depending on the quality of the respective sequence read).
  8. What does "50 bp before start of sequence" mean (comment in the T-DNA border field of the Show Sequence page)? One of the first steps of FST processing is T-DNA vector sequence detection and the subsequent removal of this sequence. If it was not detected, the T-DNA border field will show "not detected". If T-DNA sequences are detected, it will be removed, and its length will be recorded. "50 bp before start of sequence" means that at the 5' end of the original FST sequence there was T-DNA sequence detected, and that the begin of original FST is 50 bp away from the sequence shown on the web page. This information helps to deduce the insertion site of the FST.
  9. How is a "gene hit" and a "CDSi hit" defined at GABI-Kat? Since we use the A. thaliana AGI gene code (see FAQ 12) as a basis for searching and identifying insertions, we use the terms "genome hit" and "gene hit" to describe the quality of FST's. A FST is a genome hit when the sequence shows significant similarity (BLAST e-value lower than 5e-4) to the A. thaliana genome sequence. A FST is a gene hit when it is a genome hit and the expected insertion site is located between 300 bp upstream of ATG and 300 bp downstram of stop codon of an annotated gene. A FST qualifies as CDSi hit if the predicted insertion locates between ATG and STOP (CDS plus introns) of an annotated gene. Before 15.02.2003, we used an old defination of gene hit: the FST overlaps with the genomic sequence between ATG and STOP (CDS plus introns) of an annotated gene.
  10. I found several FSTs indicating an insertion at the same position, and they correspond to very similar line IDs, like 012B03, 012B04 and 012C04. Which one should I choose? Most probably this is because of contaminations that happen during the high-throughput PCR procedure. We STRONGLY suggest you request all of them. Finally we will confirm only one of the lines in such a "contamination group". You will be charged only for the confirmed line. There is no way to predict which one is true from the sequences (e.g. BLAST e value) in advance, because the FST from the real line could have a bad sequence for many different reasons.
  11. Is the insertion site of the FST in the Graphic View exact? No, the position of FSTs in the Graphic View page is not exact. For example, the indicated positions are shifted to prevent one FST from overlapping to another. The shifted distance can be up to several hundred basepairs.
  12. Which genome annotation is the basis for the current version of SimpleSearch? Our current FST annotation is based on TIGR v5 A. thaliana genome sequence and BAC annotation. We are working on an update to switch to TAIRv9 (as of Feb 2010).
  13. What does the "line availability" on the "show sequence" page of SimpleSearch mean? If the line availability is "donated to NASC" or "available from NASC (......)", the line has been donated to NASC and can not be ordered from us; please order it from NASC (see also FAQ 22). If the line availability shows "line died - no seeds available", the line is lost and should not be ordered. Reasons for losing lines are for example sterility of the T1 plant, failed germination of T2 seed, or other reasons that result in lack of offspring seed. Note that even for lines that currently display "available from GABI-Kat" for line availability, it is still possible that for example the T2 seeds do not germinate or that all T2 plants die prior to seed set. In addition, line availability does not guaranty success of confirmation, although it is a first step in this direction.
  14. What does it mean if SimpleSearch reports the "Confirmation Status" of an insertion to be "failed"? We only deliver seeds of lines if the respective insertion site has been confirmed. This confirmation is successful for about 80% of the insertions. In the remaining 20% of cases, the confirmation status of that insertion is set to "failed", and the line is not delivered. During the confirmation process, we check for DNA quality of the T2 DNA, perform a control PCR to detect the resistance gene, order a second primer when the first fails, and redo the confirmation if there are hints for technical problems.
  15. How long it usually takes to receive the ordered lines? Usually it takes about three months to finish the confirmation and to send you the seed (if confirmation was positive). Some lines have only set few T2 seed, or they show only very low germination rates. In these and other related cases, we can not use T2 seedlings to prepare genomic DNA and have to wait for adult T2 plants to harvest leaves for preparing DNA. And if the T2 seeds were used up in the confirmation, we have to wait for the harvest of T3 seeds to get something we can deliver. In such a case, you will find the status "confirmed" in request tracking, but still "in preparation for sendout" for quite some time.
  16. Why do we sometimes receive seeds very long after an order? We try to get the confirmed lines to users within three months. In some cases, it takes longer, for example when the plants grow poorly or if the controls indicate a problem that causes a repetition of the confirmation process. However, our rule is that we do not charge the line fee if we fail to deliver within 6 months. In most cases, there is no chance to confirm a line after we attempted for such a long time. But there are exceptions, which include the resolution of "contaminations" (see FAQ 10) or errors in the assignment of seed to FST. In these cases, it happens that even after two years seed is delivered. This should also to make transparent to the person who initially ordered a line that "his" or "her" mutant is donated NASC.
  17. What is the confirmation rate? The confirmation rate is about 80 % of all FSTs (FSTs were derived from DNA extracted from leaves of T1 plants, the confirmation is usually performed with with DNA extracted from several T2 plantlets).
  18. How can I check the status of my seed order? After we received all the necessary documents of your seed order, you will receive a notification e-mail which confirms that we processed your order. There is a request UID in that e-mail, and you can use this request UID to check the status of your order in the request tracking page.
  19. Could you tell me that if GK line 123A04 has been ordered by someone else before, and who? No, such information will not be disclosed.
  20. I have received the seeds with a report sheet. Can I get the sequences on the report sheet electronically? Yes, this data can be abtained from the get confirmation sequence page. You only need to enter the sequence identifier in the first line of the FASTA format sequence.
  21. The new MTA (pv20050624) with a line fee of 100,- EUR is available on your web site since 24.06.2005. What happens with line orders which were placed before that date? Unpaid invoices need to be paid according to the amount mentioned on the invoice. All invoices issued after 24.06.2005, including those for lines that were in the confirmation pipeline in June 2005, will be based on the fee of 100,- EUR per line.
  22. I am interested in getting GABI-Kat lines, but how do I know if and when these lines will be available at NASC? Please check the NASC website. Transfer of confirmed GABI-Kat lines to NASC has been initiated in June 2005. The number of lines at NASC will increase over time and you can find numbers on the GABI-Kat News & History page. In SimpleSearch, we have displayed this information for donated lines in the "line availability" field on the "show sequence" page. You can look at an example by searching for insertions in the gene At2g31180; the result is line 067H03 that has been confirmed to contain an insertion in At2g31180 and is only available from NASC.
  23. If I order a line from GABI-Kat and confirmation is successful, when is that line given to NASC? Usually we deliver T2 seeds to those who order lines at GABI-Kat. It takes another generation to harvest T3 seeds which are finally given to NASC. We need time to process and prepare the T3 seed bags for delivery to NASC, and NASC needs some time to integrate our lines into their seed store and database. As a result, you will get the line significantly before it becomes available from NASC.
  24. What happens to the executed MTAs that are sent to GABI-Kat? We start the confirmation process after we obtain MTAs which fit an order that has been placed via our web order form. After that, there are various options: (i) if there is no web order fitting a MTA that reaches us, the MTA may sit forever and nothing happens; (ii) if your MTA fits a web order, you will receive an e-mail message that the confirmation process has been started, and if the confirmation process is successful you will get back the MTA together with seed from the line(s) you ordered. To reduce administrative workload, we DO NOT send back the MTAs separately; (iii) in case that ALL lines on a given MTA fail to confirm, no seed is sent to you and therefore the executed MTA stays with us - these MTAs are send back every three to four months.
  25. I ordered lines from GABI-Kat before, and now I would like to order a few new lines. Do I need to send a new MTA for a new order? Yes, for sure you need a new MTA for other (new, additional) lines! The new line IDs (and the AGI codes or BAC IDs) need to be written on the MTA. You also need to send the corresponding purchase order and you need to submit the new data via our web order form.
  26. Do you accept payment by credit card? Yes, since September 2009 (and after years of asking for this option) we can also accept payment by credit card! You can use VISA or MASTERCARD. The procedure is as follows:
    • you download the "Credit card payment form" from the download page;
    • you print the form and fill it with the relevant information;
    • you send this filled from with your signature to us BY FAX;
    • we charge your account.
  27. What happens if my institution does not pay the line fee? If we can not observe that the line fee for (a) delivered line(s) is paid after a few months, a billing reminder e-mail is sent to the person who received the seed. If there is still no money coming in after that first reminder, a second billing reminder is sent. If there is still no response to the second reminder, the institution of the person who did not pay is "black listed". That means that we will not deliver seed to that institution any more until the case is solved. This consequence is mentioned in the second billing reminder e-mail. The third level is a "snail mail" letter to the administration head of the institution.
  28. What does the name GABI-Kat stand for? Please see this page for the answer.
  29. How is the term "T1" interpreted by GABI-Kat? We now use the following nomenclature for seeds and plants, starting with plants being infiltrated. The clarification is consistent with the paper by Ken Feldmann "T-DNA insertion mutagenesis in Arabidopsis: Seed infection/transformation" (Methods in Arabidopsis research, ed. Koncz, Chua & Schell, 275 - 289; World Scientific Publishing 1992).
    • T0 plants - the plant which is infiltrated with the agrobacteria (we mean T-zero or T-null!). These plants are setting T1 seed.
    • T1 seed - seed produced by T0 plants. These seed are in their testa genetically identical to T0 plants, but contain T1 embryos.
    • T1 plants - plants growing out of T1 seed. These plants are setting T2 seed. T1 plants are usually hemizygous for the insertion. T1 plants containing an (intact) insertion are SUL-resistant, and we are isolating DNA from leaves of resistant T1 plants for FST generation.
    • T2 seed - seed produced by T1 plants. These seed are in their testa genetically identical to T1 plants, but contain T2 embryos. This is the seed which is sent out.
    • T2 plants - plants growing out of T2 seed. These plants are setting T3 seed. T2 plants from a family segregate for the insertion(s) and for resistance. We grow some T2 plantlets to isolate T2 genomic DNA for the confirmation PCR. For lines containing one insertion locus, 33% (statistically) of the resistant T2 plantlets should be homozygous for the insertion (... you do recall Gregor Mendel' law?).
    • T3 seed - seed produced by T2 plants. These seed are in their testa genetically identical to T2 plants, but contain T3 embryos .......... In cases in which we have no T2 seed left after confirmation, we deliver a set of T3 seeds. These T3 seeds are those that are sent to NASC. A given T3 set contains seeds of all T2 plants that did produce seed (separatly for each T2 plant).
  30. How to get a probe to check for the insertion on a DNA gel-blot? We suggest that you amplify a probe from genomic DNA of the lines. We prefer PCR amplification over the preparation of plasmid DNA from large low-copy vectors.
  31. What is the difference between a single line and a T3-set at NASC? GABI-Kat lines that have been confirmed are transferred to NASC as sets of T3 seeds. Each line within a set is a sister line of the other lines in that set. The T3 seeds are harvested from individual T2 plants from a segregating family. In addition, all T2 plants were sul-resitant. As a result, the T3 set has a high probability to contain seeds from at least one homozygous T2 plant for a given insertion line.
  32. What if my question is not answered in the FAQ? E-mail us at info()gabi-kat.de Make sure to use an informative subject header line. Mails that have no subject or include a subject text that looks like SPAM will not be read - in fact, such emails are deleted automatically. 
  33. I wrote e-mail to GABI-Kat and I always got reply e-mails with something like GKT:XXXXXXXXXX or CBT:XXXXXXXXXX in the subject line, what's that? Since August 2003, we used an email management system (OTRS, http://otrs.org/) to keep track of all the email to and from GABI-Kat. The GKT:XXXXXXXXXX or CBT:XXXXXXXXXX is the identifier of the thread, please do not modify it when you reply or forward.
    UPDATE: the OTRS system was discontinued in August 2008 and replaced by a simple shared IMAP mailbox. The GABI-Kat Team takes care about answering incoming questions.  
  34. How should I cite GABI-Kat in my publication? Please see this page for the answer.
Last Updated on Wednesday, 28 April 2010 14:02