Kafatos:Ordering

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Ordering Information

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Primers We order primers from Sigma-Genosys Please use this Excel template and email as an attachment to Pove or Yasmeen Orders require the following information: Name (35 characters fit on the tube label) Sequence PDF with detailed instructions and modification codes Scale 0.025, 0.05, 0.2, 1, 10 and 15 μmol 0.025 must be between 12 and 35 bases and can only be desalted Purification DST (desalted), RP1 (reverse phase cartridge), HPLC and PAGE

DNA Sequencing Sequencing done by The Natural History Museum Contact: Julia Llewellyn-Hughes for info on sample requirements and to schedule a drop-off time. Tel: 0207 942 5352 Fax: 0207 942 5347 Get a PO Number from Pove or Yasmeen. The cost is £8/reaction. Complete this form and make a copy. Contact Julia to schedule a drop-off time Drop off location - The Bronze door of the Natural History Museum Primers Common primers: T3, T7 etc are supplied by the facility. Custom primers: Submit 10 μl of a 10 μM stock to the facility. Templates Small PCR products: 10 μl of Qiagen PCR purified DNA at 100 ng/μl is sufficient. You must specify the length of PCR products. This is not necessary for plasmid templates. Vector templates: 10 μl of Qiagen prep in water. They use 100 ng per reaction. The lower limit for concentration is 20 ng/μl. Submit the concentrations, however, they will confirm it in the facility. Other DNA? Please update with information if you enquire about other templates. Submitting DNA samples for sequencing: (Imperial College) Wanda Stow (3rd floor) ! We have in the lab, stocks for T7, SP6 and M13f and M13r sequencing primers (ask Yasmeen)) Mix one template and one primer in a tube, taking care to follow guidelines on template preparation and quantity. We prefer that you use 0.2l or 0.5l tubes, or for large numbers you could use 96-well plates. Label each tube clearly using your initials and sequential numbers from 1 to..xx. We are processing large numbers of samples daily, so this helps to streamline the process - if we receive any other labelling format, the samples will be assigned new labels! Protect your tubes appropriately if they are to be posted, and ensure lids are properly sealed to avoid evaporation - a little parafilm will help. Please do not leave tubes loose in an envelope, or tape them to your paperwork! Instead place in a small sample bag (or bags). Please follow the guidelines below for primer design, template preparation and optimal concentrations. If you have successfully performed sequencing reactions elsewhere with different concentrations, feel free to use them but be aware that we will add a 6l volume of whatever mixture you supply to the sequencing reaction (4µl for GC-rich, cosmid or lambda templates or 12µl for BACs). Please adjust concentrations accordingly! All sequencing samples must be accompanied by a completed Sequencing Request Form, DNA Risk Assessment Form and a Data Sheet. Please keep a reference copy of the data sheet if necessary. In addition each order must be accompanied either by a signed and stamped GL/POETA Journal (Imperial College customers) or a valid purchase order (external customers). On the day we receive samples, you will receive an email acknowledging receipt. The reactions are performed using Applied Biosystems BigDye version 3.1. The cycling is done overnight using one of four optimized protocols appropriate to the nature of the template provided and the Tm of your primer. The following morning the post-reaction cleanup is performed to remove unincorporated dye-terminators - we use an ethanol and sodium acetate precipitation procedure. The dried pellet is re-suspended in deionised formamide once there is an available slot on the sequencers. Template preparation Always take time to purify and quantify all types of template DNA accurately, as this really does have significant effects on the success of automated sequencing reactions. Use clean-up kits designed for fluorescent automated sequencing. Systems that we have found work well include: -Qiagen mini-preps - perform dilution/elution steps using water rather than the buffer supplied -microCLEAN from Microzone -Templiphi from Amersham Pharmacia - an enzymatic amplification technique for generating template copies Determine concentrations by checking OD rather than running gels. Check the 260/280 ratio - anything less than 1.5 may indicate protein contamination. Ensure complete removal of PCR primers to avoid multiple sequencing. Primer design The optimal primer length is 18 bases (although we find slight deviations from this are usually OK). Optimal GC content 50% Optimal Tm is 55°C (range from 48° C to 65° C will usually work fine). If the primer you are using has a Tm lower than 48°C and you are unable to increase it (e.g. by adding bases) it may be useful to double the usual recommended concentrations, keeping the final volumes the same. Template and Primer Concentrations (The following apply to standard templates only - please see below for GC-Rich Templates, Cosmid and Lambda). For Double Stranded Plasmid DNA: Supply 1µg template DNA mixed with 12.8pmol of primer made up to a total volume of 12µl with deionised water. For Single Stranded Plasmid DNA: Supply 200ng template DNA 12.8pmol of primer made up to a total volume of 12µl with deionised water. For PCR Products: The amount you send is dependent on the size of the product: 200-600bp products: supply 80ng template DNA and 12.8pmol primer, made up to a total volume of 12µl with deionised water 601-1000bp products: supply 120ng template DNA and 12.8pmol primer, made up to a total volume of 12µl with deionised water 1001-1500bp products: supply 300ng template DNA and 12.8pmol primer, made up to a total volume of 12µl with deionised water 1501-3000bp products: supply 400ng template and 12.8pmol primer, made up to a total volume of 12µl with deionised water >3000bp products: treat as plasmid TEMPLATE and primer concentrations for GC-Rich Templates, Cosmid and Lambda. For GC-Rich Plasmid DNA : Supply 4µg template DNA (or 800ng for single stranded plasmid) mixed with 12.8pmol of primer made up to a total volume of 8µl with deionised water. For GC-Rich PCR Products : The amount you send is dependent on the size of the product: 200-600bp GC-Rich products: 160ng template DNA and 12.8pmol primer, made up to a total volume of 8µl with deionised water 601-1000bp GC-Rich products: 240ng template DNA and 12.8pmol primer, made up to a total volume of 8µl with deionised water 1001-1500bp GC-Rich products: 600ng template DNA and 12.8pmol primer, made up to a total volume of 8µl with deionised water 1501-3000bp GC-Rich products: 800ng template DNA and 12.8pmol primer, made up to a total volume of 8µl with deionised water >3000bp GC-Rich products: treat as GC-Rich plasmid Cosmid or Lambda templates Supply 8µg template DNA mixed with 25pmol primer made up to a total volume of 8µl with deionised water. BACs: Supply 4µg template DNA mixed with 50pmol primer made up to a total volume of 25µl with deionised water. Half of the mixture you supply will be used for the sequencing reaction and the other half is stored in case further work or a repeat reaction is required. In order to streamline the processing of large sample numbers we make no adjustments to concentrations at this end, so we would ask that you ensure your mixture contains the recommended amounts of template and primer.

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