Nevada Genomics Center - Sample Submissions

Sequencing

The samples should be labeled with the order number and an individual number for each tube.  Do not try to write long sample names on the tubes.  Please read our guide on the dnaTools server for instructions on submitting sequencing sample requests to the NGC:

Sequencing Sample dnaTools Instructions

Quantity

The amount of template DNA and primer required dependant on the size of the molecule. The formulas below are a guide for arriving at the best amount to use.

Templates

Primers

Sample Submission Format and Volume

It is essential to set-up the sequencing reactions with the correct amount of template and primer (see guidelines above).  Therefore, it is important to accurately quantitate the DNA.  Once you have a routine method working, it’s possible to just estimate the DNA based on previous runs.  We can quantitate DNA samples by fluorimetry, using PicoGreen, if you do not have access to a reliable instrument for quantitation.   If you wish to submit samples for quantitation, please send the template DNA and primer in separate tubes so that the proper amount of primer may be added to the correct amount of template following quantitation.

Our PCR clean-up will remove any unused primers, dNTPs, and salts.  If samples are submitted for PCR clean-up, please send an aliquot of the sequencing primer(s) in separate tube(s) so that the primer may be added following the clean-up and quantitation. 

When setting up you sequencing reactions, begin with template and primer that are in deionized sterile water.  Never set-up sequencing reactions with template or primer that are in TE or a similar buffer!   The EDTA present in many buffers will bind the magnesium that the Taq enzyme requires for activity and thus the EDTA will hinder the sequencing reaction.  Upon arrival at NGC, all samples are dried down and resuspended at 5ul, so any salts or buffers present in your sequencing sample set-up may be concentrated; therefore it is essential sequencing samples are in deionized sterile water.  Your sample volume is not critical; however, we recommend the volumes be at least 5ul and less than 40ul.

All samples must be submitted in either 0.2 mL 8-strip PCR tubes or 96-well plates.  Strip tubes are 8 tubes that are connected together, and you can remove the extra tubes if you do not have 8 samples in the strip.  The maximum number of samples in a 96–well plate is 95, as 1 well must be left for the facility's internal run control.  To qualify for the full plate rate samples MUST be a 96-well plate appropriate for use on a thermocycler.  You may also submit less than 96 samples in a plate, but you MUST arrange your samples by COLUMNS (A1-H1, A2-H2, etc.), not by rows (A1-A12, etc.).  Do NOT leave any empty wells between samples; all unused wells must be consecutive at the end of the plate.  Please add a note to your job request listing the wells that are empty.  Label your strip tubes or 96-well plate with your dnaTools job order number.  On the strip-tubes label each tube with the sample number from your job submittal.  The number marked on each tube of the strip tubes must correspond to the sample number on the sequencing order form.

Bacterial Sample Submissions

NGC can not accept any pathogenic E. coli or strains carrying toxin genes.  Our MOUA (Memorandum of Understanding and Agreement on Use of Biological Agents and Recombinant DNA Research) with the University of Nevada, Reno limits us to working only with CDC and NIH designated Biosafety Level 1 organisms and Risk Group 1 recombinant DNA.  Biosafety Level 1 organisms and Risk Group 1 recombinant DNA are described by the CDC and NIH respectively as, “not known to consistently cause disease in healthy adults.”   No routes of infectivity or routes of transmission are given by CDC or NIH and both are considered low risk.  If you are submitting E. coli to us, please list the E. coli strain, its Biosafety Level, and the vectors that the E. coli carries.  By UNR restrictions, any research lab at UNR submitting recombinant DNA for sequencing must have a valid MOUA for work with recombinant DNA on file with the Institutional Biosafety Committee. 

Please contact us if you plan on submitting bacteria for plasmid isolation.

Fragment Analysis

Please read our guide on the dnaTools server for instructions on submitting fragment analysis sample requests to the NGC:

Fragment Analysis dnaTools Instructions

We prefer that you have a robust PCR reaction which requires considerable dilution in order to be on-scale for the 3730 run so that most of the PCR reagents are diluted out.  If the PCR products being run need less than 1/100 dilution, we recommend an ethanol precipitation prior to sending the samples for 3730 analysis so that the excess PCR reagents are removed.  If these low dilution samples are not cleaned up we can not guarantee the success of the 3730 run.  We will run dilution test samples for you to determine the best dilutions before running a plate of samples.

We can also run the PCR reactions for you but the list price does not include the price of your labeled primer.  If we are to run the PCR reactions you must have optimized the conditions for your primers.  If you do not multiplex the PCR reactions but wish to multiplex for the 3730 run, please note that the price for multiplexing is for the dilution and addition of one PCR reaction to the 3730 plate, so multiply this by the number of labeled primers you wish to multiplex.