Kafatos:Ordering: Difference between revisions
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===Primers=== | ===Primers=== | ||
We order primers from [http://www.sigmaaldrich.com/Brands/Sigma_Genosys.html/ Sigma-Genosys]<BR> | We order primers from [http://www.sigmaaldrich.com/Brands/Sigma_Genosys.html/ Sigma-Genosys]<BR> | ||
Please use this [[Media: | Please use this [[Media:Oligo_order_form3.xls | Excel template]] and email as an attachment to [[Kafatos:Povelones%2C_Michael | Pove]] or [[Kafatos:Ghani%2C_Yasmeen | Yasmeen]] | ||
Orders require the following information: | Orders require the following information: | ||
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===DNA Sequencing=== | ===DNA Sequencing=== | ||
Sequencing done by | Sequencing done by Beckman Coulter Genomics | ||
* £4.50 per reaction | * £4.50 per reaction. | ||
* You'll need 2 reactions per sample (forward and reverse) | * You'll need 2 reactions per sample (forward and reverse) | ||
* <font color="red">NEW!</font> [[Media:Beckman_Coulter_Genomics_template.xlsx | Download this form]] and fill it in. Print it and submit it with your samples.<BR> | |||
* <font color="red">NEW!</font> [[Media: | |||
* Service = PE | * Service = PE | ||
* Check Email text file | * Check Email text file | ||
* Once you've submitted the order, | * Once you've submitted the order, Beckman will email you a copy of this form - Print it out to submit with your samples. | ||
* Plasmid DNA - ideally 10 μl of 100 ng/μl | * Plasmid DNA - ideally 10 μl of 100 ng/μl | ||
* Clean PCR product - ideally 5 μl of 50 ng/μl | * Clean PCR product - ideally 5 μl of 50 ng/μl | ||
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* Primers - either universal (T7, T3 etc.) or user supplied (provide 10 μl of 10 μM primers) | * Primers - either universal (T7, T3 etc.) or user supplied (provide 10 μl of 10 μM primers) | ||
* Wrap samples in parafilm and seal in a plastic bag with your order form. | * Wrap samples in parafilm and seal in a plastic bag with your order form. | ||
* Take to reception and place in the DHL bag labelled | * Take to reception and place in the DHL bag labelled Beckman on the mail room table. | ||
* Collection time is 3.30pm. | * Collection time is 3.30pm. | ||
* The service is a one-day turnaround so you should have results by email the day after they receive them. | * The service is a one-day turnaround so you should have results by email the day after they receive them. | ||
* If required, | * If required, Beckman can also synthesize a sequencing primer for your sequencing reactions (this will take one day longer before you get your results) | ||
Latest revision as of 11:13, 14 June 2011
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Ordering Information
For Lab Orderers |
PrimersWe order primers from Sigma-Genosys Orders require the following information:
|
DNA SequencingSequencing done by Beckman Coulter Genomics
Sequencing done by The Natural History Museum
! We have in the lab, stocks for T7, SP6 and M13f and M13r sequencing primers (ask Yasmeen))
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.
-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
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).
Template and Primer Concentrations (The following apply to standard templates only - please see below for GC-Rich Templates, Cosmid and Lambda).
Supply 1µg template DNA mixed with 12.8pmol of primer made up to a total volume of 12µl with deionised water.
Supply 200ng template DNA 12.8pmol of primer made up to a total volume of 12µl with deionised water.
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.
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.
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
Supply 8µg template DNA mixed with 25pmol primer made up to a total volume of 8µl with deionised water.
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. |
Poster PrintingPlease visit the Imperial College poster printing core facility for instructions |