FAQs - Frequently Asked Questions

Haplobank Cell

  • How do we handle ES cell lines?

    Basically as you would handle any other (ES) cell line, standard cell culture practice/techniques apply. Please check out the protocol section for general guidelines as to how the cells should be handled. For a general description, please check the following publication: Elling et al. (2011). Cell Stem Cell, 9(6): 563-74. If you are interested in doing a screen with one of our ESC libraries, we would be happy to offer you a collaboration. You would then also have the possibility to get a general ES cell culture training.
  • Which serum do you use?

    We usually do a serum test assuring that we select a batch which allows for rapid proliferation and a high rate of colony formation without massively inducing differentiation of our ES cell lines. We usually test around 5-6 different batches, depending on availability. You should do the same and test a serum on any ES cell line which you may have available at your institute. Alternatively, you might want to use the serum that the stem cell facility at your institute suggests/uses successfully on their ES cell lines.
  • What should I use as control cell line in my experiments?

    The best control cell line would be a non-disruptive sister clone. These can be purchased in the “Cell Information” by clicking on the “cell ID”. You can also browse our protocol section on how to generate such a “flipped” sister clone by yourself. Our wildtype cell line “AN3-12” can function as a control cell line as well. If you want to order AN3-12 additionally, search for them in the catalogue, add them to the shopping cart (office@haplobank.at) and don’t forget to add them in Annex 1 on the MTA.
  • In which format do you supply the parental ES cell line AN3-12 and clones from your collection?

    We provide the parental cell line AN3-12 in 2D Matrix tubes in a density, which is high enough to seed them directly onto a 10cm cell culture dish. Likewise, clones from our collection are frozen down into 2ml cryo vials, which contain also enough cells to seed onto a 10cm dish.
  • What do the annotations in the column "Mutation" mean and what kind of mutation should I choose?

    The column “Mutation” provides information about the site of mutagen integration relative to the gene body:







    Due to several transcripts per gene an insertion can have several of these features. If several feature types are assigned to an insertion following priorities are recommended to be applied (S = sense, AS = antisense):
    1 CDS_S
    2 CDS_AS
    3 5UTR_S,
    4 5UTR_AS
    5 1Intron_S
    6 Intron_S
    7 ncExon_S
    8 ncExon_AS
    11 3UTR_S
    12 3UTR_AS
    13 1Intron_AS
    14 Intron_AS
    15 Upstream_S
    16 Upstream_AS
    17 InterUp_S
    18 InterUp_AS
    19 InterDown_S
    20 InterDown_AS

    We regard a mutation as disruptive, if the mutagen is inserted in 1Intron, Intron, 5"UTR, CDS, and ncExon. We would recommend going for an insertion in the first Intron (1Intron) if possible. Clones that carry the gene trap in an intergenic region (i.e. annotated as Upstream, InterDown, InterUp), cannot be regarded as knockouts of the respective gene. Please also keep in mind that a mutation in the coding sequence is phenotypically not reversible.
  • What quality controls were done before you shipped the clones?

    Every clone we expand, we routinely test for Mycoplasma-contamination using a qPCR assay and only Mycoplasma-free clones get shipped.
    We also perform a Barcode PCR and an Integration PCR to confirm the correct identity and the correct genomic integration site of the clones.
    For inverted sister clones, we also check the successful inversion of the gene trap.
    We provide you with a summary sheet of all quality control results when we ship the clones.
  • Where is the genetrap located relative to the "mapped sequence"?

    That depends on the mutagen: in Retro and Lentiviral clones the genetrap lies downstream of the mapped sequence, in Tol2 clones upstream.
  • What does it mean if Sanger Sequencing of the bcPCR product gives more than one Barcode for a clone?

    Either the clone carries more than one genetrap (i.e. white clones) or the clone is a mixed clone, i.e. it contains another clone, which could have been introduced during the production process. Compare the results to the intPCR and keep this in mind when working with this clone.
  • What do I do if the intPCR is not working and how do I interpret deviations from the expected pattern?

    First make sure that you chose the right combination of primers and DNA (see intPCR protocol) and if possible try it again with the other primer combination (black ones in the combination overview). If you get no PCR product at all (not even in the purple lane) consider using another polymerase, doing a new gDNA prep or ordering new primers.
    If you only get a PCR product in the purple lane compare the results to the bcPCR and consider performing an independent iPCR.
    If you get the expected PCR products (orange lane and purple lane) and an additional product in the green lane, showing the same size as the product in the purple lane, the clone can be considered as heterozygous and is no true knockout. You should compare the results to the bcPCR and consider ordering an other clone (if available).
    If you get PCR products only in the green and purple lane (same size) the clone is also potentially heterozygous. You should do an iPCR and also compare the results to the bcPCR.
  • Do the cells stably maintain their haploid phenotype?

    No, our haploid ES cells become diploid over time, i.e. the longer you keep them in culture, the more cells become diploid. It was estimated that ~2%-3% of haploid cells become diploid for each day in culture. Check the protocol section on how to sort for haploid cells.
  • Does diploidization influence the knockout efficiency of targeted genes?

    No, as clones were mutagenized at their haploid state for which we select for by FACS before and after mutagenesis. Thus, diploidization is not a problem because the insertions will be present in both "alleles". However, there is a chance that we mutagenized a diploid cell and it escaped our selection against diploidy. In this case, the cell line was not homozygously targeted and is not a knockout. This can be verified by performing the Integration PCR (see intPCR-protocol in the protocol section).
  • What tests should I run after receiving my clone-of-interest?

    After successfully reviving, expanding, and preserving the clone at your lab, we recommend doing some quality controls yourself. Firstly, you might want to start with the bcPCR to confirm that you in fact received the correct clone, which you do by simply amplifying the internal barcode of the clone at hand and comparing it to our database. Secondly, you can do an iPCR to retrieve the integration site(s), and/or you can design primers around the integration site and do the intPCR. The integration primers in combination with the mutagen-specific primers will only give you a PCR product if targeted correctly. Please browse our protocol section for the protocols and more details for the different PCRs.
  • How do I best confirm that a clone is a true knockout for my gene of interest (GOI)?

    We suggest that you do two things: firstly, you quantify the message of wildtype and mutagenized clones for your GOI by qPCR, respectively. Comparing both samples, you should see a massive drop in expression. Secondly, you run a Western blot for your GOI on both samples. You should see a complete loss of protein in case it is a true knockout. As for qPCR, you might want to design your primers in exons that flank the mutagen, e.g. if your clone carries the gene trap in the first intron, you would design your qPCR primers in Exon1 (fwd) and Exon2 (rev), if possible. Should you not be able to confirm the knockout of your GOI by both methods, please get in touch with us at office@haplobank.at to discuss options.

Shipment

  • How long does it take to get the ES cells clones?

    It usually takes us 4-6 weeks to expand the clones and do the quality controls. If you ordered the inverted sister clones it will take us 6-8 weeks. You will get an E-Mail notification when your clones are ready for shipment. Once the MTA and all clones are ready (clones are expanded and QCs are done) they get shipped on the following Monday.
  • I would like to order ES cell lines. What are the shipping costs by express courier?

    We use a transport box which accommodates a plastic bag with clones and fill it up with 5-6 kg of dry ice, i.e. we use the same pack and dry ice amount for any cell line shipment regardless of the cell line quantity. The estimated FedEx shipment fees to the USA would be around € 270, and around € 150 for destinations in Europe. Please contact your local delivery service for detailed information about shipping costs.
  • How long does the shipment take?

    In Europe, the package is delivered within 2 working days. Shipments outside of Europe usually take a few days longer e.g. Japan 2-3 days, USA/Australia up to one week. Please note that we only ship cells on Mondays, regardless of the destination.
  • ES cell lines were destroyed during the shipment. Can you resend them?

    If you notify us within one week we will resend the clones you ordered free of charge, but you will have to pay the shipping costs.
  • Do I need an import permit to get the ES cells?

    Please inquire with your local authorities and make sure that we have all required documents in place for successfully shipping the cells to your country. This may also require special courier services as e.g. FedEx may not ship a dry ice parcel to any country. Should your country require special permissions to import murine ES cell lines (or any cell line for that matter), please make sure that you have all required documents in place. The same holds true for shipping labels, should they be required. Haplobank will not replace cell lines, which have been refused at the customs because of missing or expired importing documents, and will likewise not cover any costs associated with such a delay in delivery.
  • Can the shipping address be different from the address on the import permit labels?

    It is not recommended that you borrow and provide us with the shipping information from a colleague, because this might cause confusion at the customs (or import regulation agency) and result in a massive delivery delay. In this case Haplobank will not replace any cell lines which did not survive the shipment.
  • Why do you use only DHL/FedEx and not postal service?

    We have a long and good experience with these courier services and also most researchers have their own accounts set up with them.
  • How do I know when the ES cell clones will arrive?

    The week before we ship the clones we will get in touch with you to ensure that you will be there to receive them. After we shipped the clones we will send you the tracking number by e-mail.
  • Is it possible to combine several orders in one shipment?

    Yes, but only if they have been placed in the same week, if we already have the extra MTA for those clones in place, and if they don't exceed more than 100 ES cell clones in total.

Order

  • How long does it take to approve my account?

    It takes around 2 working days.
  • What information is required for the MTA?

    We require you to fill in Annex 1 of the MTA, in which you please provide the Cell ID of the clones you ordered. As for unmodified, wildtype cells, please use AN3-12 as the Cell ID. In Annex 2, we would kindly ask you to concisely describe the experiments for which you want to use the ES cell lines. Please make sure that the MTA is fully signed, i.e. by a legal representative of your institute, and you or your boss as the recipient scientist. Once you have filled in the MTA and obtained all required signatures, please send a pdf copy to to Denise Langer (denise.langer_AT_imba.oeaw.ac.at). We strongly suggest sending us a fully signed MTA as a pdf per email as opposed to a hardcopy, as this saves quite some time. Once we received your MTA, we will collect required signatures on our side (usually within 2-3 business days) and forward a pdf copy of the fully executed MTA.
  • I have created a Haplobank account a while ago. My account is still not active. Why?

    Maybe something didn’t work the way it should. If your account is not active after 3 working days, please send an email to office@haplobank.at.
  • Searching the database, I found several clones for my gene-of-interest. How do I decide which clone(s) I should order?

    There are several things that you might want to take into consideration: we have clones in our collection that either contain only one gene trap (i.e. blue clones) and clones with more than one insertion of gene trap cassettes (i.e. white clones). Should you have the option of choosing between blue and white clones, we would recommend to always go for the blue clones, as multiple insertions might mask the phenotype which you are interested in. Then you have to make a decision whether or not you want to start with a clone in which the gene trap cassette is inserted in "sense" or "antisense", and is thus disruptive and non-disruptive for your GOI, respectively. This information is indicated in the column "Rel. Orientation". As all our gene trap cassettes can be inverted, it is probably not a big concern at the beginning (see protocol in protocol section). Next you might want to decide for what kind of mutation you go for. We regard a mutation as disruptive, if the mutagen is inserted in 1Intron, Intron, 5'UTR, CDS, and ncExon (specified under column "Mutation"). We would recommend going for an insertion in the first Intron (1Intron) if possible. Clones that carry the gene trap in an intergenic region (i.e. annotated as Upstream, InterDown, InterUp), cannot be regarded as knockouts of the respective gene. Please also keep in mind that a mutation in the coding sequence is phenotypically not reversible! The choice of the mutagen is then the last thing you want to consider. If you can choose between Tol2, Tol2GT, Retro or Lenti (specified under the column "Mutagen"), you should keep in mind that Tol2GT/Retro/Lenti are enhanced gene traps while Tol2 is a polyA-trap that carries EGFP, which might be an important thing for you to keep in mind. Please check the relevant publications for details on each of the systems (Mayasari NI, Mukougawa K, Shigeoka T, Kawakami K, Kawaichi M, Ishida Y., Nucleic Acids Res. 2012 Jul;40(13):e97 and Schnütgen F, et al., Nucleic Acids Res. 2008 Nov;36(20):e133).
  • I have ordered ES cell lines some time ago. Can I check the status of my order?

    Yes, please login into your Haplobank account and click on the button Order History placed in the upper right corner on the main page.
  • I have not received any notification about my order. Is there any problem?

    As long as you got an order confirmation and sent all necessary documents (MTA, import permits, shipping labels, ...) there is usually no problem. Your order is still in progress and due to our preparation schedule it takes up to 6 weeks until your order is ready to ship. Please check the Order History on the Haplobank website.
  • I placed an order yesterday and forgot to order another ES cell line. Can I add this cell line to the existing order or should I place a second order?

    Unfortunately, we cannot modify an existing order. Within 24h you can cancel the order with full refund and place another order with additional (or less) ES cell lines. You can also place another order (only within the same week) and send us an email saying to combine the shipment of both orders. This is possible as long as the orders don’t exceed the limit of 100 ES cell lines in total.
  • Can I replace some ES cell lines, which are still pending with other ones?

    Unfortunately, we cannot modify an existing order.
  • Is there a way to order the ES cell lines and save the content of the shopping cart?

    Our website doesn’t offer a shopping cart management, but we recommend to store the Cell IDs in a list and order the list at a later point in time.
  • Can I come and pick up the cells directly at Haplobank?

    Yes of course, after placing the order, please send us an e-mail and notify us about it. As soon as your order is ready to ship, we will contact you and make an appointment for picking up the cell lines.

Payment

  • Can I get a discount price if I order many ES cell lines?

    No. Please see our payment information.
  • My credit card is valid but was rejected during the payment process. Why?

    We have no possibilities to get detailed information about user transactions from financial institutes due to security reasons. Therefore, we recommend you to try again later and if it doesn’t work to check with your bank.
  • During the payment with my credit card the system asks for a password. What password?

    Your credit card is activated with the "Verified by Visa" or "MasterCard SecureCode" service which improves the security of online payment transactions. Therefore, you need a password to verify your credit card transactions. Please contact your bank.
  • I do not have a credit card. Can I pay in some other way?

    No. We accept payment only by credit card.
  • What is a VAT Number in the registration form?

    The Value Added Tax, or VAT, in the European Union is a general, broadly based consumption tax assessed on the value added to goods and services. Every European Institution needs a VAT number for tax purposes.
  • I did not receive all ordered ES cell lines and my credit card was already charged. Will I get a refund?

    Yes of course, you will get full refund for all the cell lines we could not deliver. You will get notified about it and your credit card will be automatically refunded.
  • I do not have the invoice/credit note for one of my orders anymore. Can you resend it?

    For your convenience, you can download the invoice/credit note in your Haplobank account. Please follow these steps:
    1. Login with your user name / password
    2. Select Order History on Haplobank website and you will get a list of your orders
    3. Click on the ’View’ button to display an order.
    4. 4. Click on the ’PDF’ button beside the invoice ID to download the invoice.
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