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Department of Biology


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Frequently Asked Questions

What types of DNA templates can be sequenced?

What should the concentration of my DNA template be?

How much DNA template should I send?

Do you have primers in stock?

How much primer should I send, and what concentration?

Where do I send the samples?

Can I drop off my samples?

How much do you charge?

Do I get charged if the reactions fail?

When do I get my results?

How do I know when the results are ready?

How do I get my results?

How do I view my results?

Can I add permission for my students to access my folder on the server?

I think the core messed up my sample.

My sequencing failed-why?

 

What types of DNA templates can be sequenced?

 

DNA sequencing can be performed on PCR products, plasmid prep DNA, cosmids or BAC.

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What should the concentration of my DNA template be?

 

In general, the concentration of double stranded DNA under 10Kb should be around 200 ng/ul, and rom 10Kb – 20Kb around 400 ng/ul.  PCR products should be around 20 ng/ul.  Please click here for more DNA sample guidelines.

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How much DNA template should I send?

 

For double stranded template of 200 ng/ul and PCR products of 20 ng/ul, send 10 ul for each reaction.  For more specific guidelines, please click here.

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Do you have primers in stock?

 

Yes, we have pUC/M13 forward and reverse primers, -20 M13 forward, T7, T3 and SP6 primers.  For the sequence of the primers, please click here.

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How much primer should I send, and what concentration?

 

Primer concentration should be 5 uM (which is the same as 5 pmol/ul), and 5 ul per reaction should be supplied.  For more specific sample guidelines, please click here.

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Where do I send the samples?

 

Submit samples with the proper form to:

Genomics Core Facility

Department of Biology

Mail Stop 551

ATTN:  Denise Mayer

 

The request forms can be found here.

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Can I drop off my samples?

 

Yes, you can drop off samples to Denise Mayer in N201, or leave them in her mailbox in N113.

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How much do you charge?

 

Sequences are $8.00 per single template/primer reaction for University researchers, and $10.00 per single template/primer reaction for non-University researchers.  Primer Extension is $4.00 per sample for University members, and $6.00 for researchers outside the University. Please check the Genomics Core Facility policy page for other information.

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Do I get charged if the reactions fail?

 

If a reaction fails due to poor template, the researcher will be charged for that reaction.  A re-run can always be requested, and we will do our best to help you troubleshoot a difficult template.  If the error turns out to be a result of the Core Facility, the re-run will be free of charge. Please check the Genomics Core Facility policy page for other information.

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When do I get my results?

 

Your results are ready withing 3 – 4 working days, depending on workload. Please check the Genomics Core Facility policy page for other information.

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How do I know when the results are ready?

 

You will be informed by email when your results are posted to the server.

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How do I get my results?

 

Sequence results are posted to the server, in folders assigned to the individual researchers and their students.  For directions on how to access the folders on the server, please click here.

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How do I view my results?

 

The text file, with just the sequence, can be viewed in Word or any text program.  The electropherogram can be viewed in several different freeware programs.  Information on these programs may be found here.

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Can I add permission for my students to access my folder on the server?

 

Yes, send an email to Denise Mayer to request the student be added to your folder.  Be sure to include the student's PirateID in the email.

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I think the core messed up my sample. 

 

If your sequencing failed, and you think it should have worked, you can request the Core Facility to re-run the sample.  We will set up the reaction again using the same tube of template and primer.

If the sequencing works, you will not be charged for repeated reaction.  If the sequencing fails again, you will be charged for both the original and repeated sequencing.

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My sequencing failed-why?

 

The most common reason for failed sequencing is template problems.  Make sure you quantify your DNA-don't just write down the concentration we request.  When you quantify your template by spectrophotometry, check the 260/280 ratio.  The ratio should be around 1.8-a significantly lower value could be due to phenol or RNA contamination.  Verify the purity visually by running the template on an agarose gelm making sure there is only one band in the sample. Other problems could be multiple priming sites, inaccurate calculation of primer concentration, secondary structure difficulties or resuspending the template or primer in a solution containing EDTA.