Central Services Core

Nevada Genomics Center - Sample Submissions

Sequencing

The samples should be labeled with your initials, 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.

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 thermocycler plate.  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.).  Label your strip tubes or 96-well plate with your dnaTools job order number, and 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.

Sequencing samples with template and primer must be in deionized sterile water or completely dried down.  Upon arrival at NGC, all samples are dried down and resuspended in the appropriate volume, so your sample volume is not critical.  However, we recommend the volumes be at least 5ul and less than 40ul.  Do not use TE because EDTA's ability to bind Mg++ can hinder the sequencing reaction.  

Dried samples need only to be covered (tape, tube caps) for shipping.  If samples in solution are mailed to us overnight, no refrigeration (ice packs, dry ice) is required.  Tape seals are not adequate to prevent leaking in 96-well plates that are shipped. We recommend using strip caps and sandwiching the plate between two tip insert racks and securing with tape as shown in the picture below.  Be sure that the tips of the tubes are not protruding through the bottom or they will likely get crushed.  Strip tubes with caps also need to be sealed with parafilm.  A little parafilm stretched tightly goes a long way and seals much more effectively than a lot of parafilm that has not been stretched out.

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 plasmind 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 which are being multiplexed need less than 1/100 dilution then we will only run them after an ethanol precipitation.  We will run dilution test samples for you to determine the best dilutions are 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.

Plates that have been setup by you and are being mailed to us, can be sent dried down by overnight delivery without ice, or if not dried down, on wet ice.  Please see the guidelines for shipping sequencing samples for packaging instructions. We will add the MW markers to samples that have been dried down