Capillary DNA Sequencing & Genotyping

Sample Prep

Single tube submissions

The GGF uses BigDye ver. 3.1 for all sequencing.  For optimal sequencing, BigDye ver. 3.1 requires 5 ng of template per 100 bp of product length.  We will use 1 µL of template provided, unless specifically requested to do otherwise.  Thus, users should adjust the concentration of product to be 5 ng/µL x product length in bp/100bp.  For example, a PCR product of 500 bp should be provided at: 5 ng/µL x 500bp/100bp = 5 ng/µL x 5 = 25 ng/µL.  As a second example, a 1000 bp PCR product should be provided at 50 ng/µL.  Plasmids are under the same constraint as PCR products where a 5kb plasmid should be submitted at a concentration of 250 ng/µL.  If your plasmids are substantially large (~10K), then some optimization may be required. 

To allow quality control assessments and sequencing, as well as re-runs where needed, we require a minimum of 20 µL of template at a concentration of 5 ng/µL per 100bp in length.  If primer-walking with the same template, please submit an extra 5µl per reaction. Samples should be diluted in high quality molecular grade water rather than TE.  Templates submitted at volumes or concentrations too low (e.g., less than 50% of the required) will not be accepted.

Plate submissions

We can sequence 96 well plates of purified products with one or two primers used for all of the wells (e.g., a plate of 96 plasmids sequenced with a forward and/or a reverse primer).  Please follow the guidelines for volume and concentration of single tube samples.  Note, however, that many users obtain satisfactory results by determining an average concentration of samples with a plate and using a single constant volume based on that concentration.  Plates that contain 19 or less samples will be charged as single tubes (e.g., 10$ per reaction).  For those plates that contain 20 or more samples (and are using the same primer set for all samples), they will be charged as if it were a full 96 well plate (flat rate of 96 x 2$ per reaction = $192.00).  Thus the more templates that you put in the 96-well plate the cheaper it becomes. As this service is provided at a large discount, we need to use automated pipetting procedures.  Thus when submitting samples in plates for cycle sequencing, please use a v-bottom 96 well polystyrene micro-titer plates rather than PCR plates. Optimal plates can be obtained from Thermo Scientific (Fisher Sci. #12-565-436) or USA Scientific (# 1833-9600).

Sequencing Primers

We have the following primers on hand that we will supply for sequencing if so desired. Please verify that the sequence of the primer listed below is the one that you need. Where custom primers are needed, primers should be provided at 3.3 µM diluted in high quality molecular grade water.

Primer Name Length Sequence
M13-20 FORWARD 18 TGT AAA ACG ACG GCC AGT
M13-48 REVERSE 24 AGC GGA TAA CAA TTT CAC ACA GGA
T7 PROMOTER 20 TAA TAC GAC TCA CTA TAG GG
T7 TERMINATOR 21 TTA TGC TAG TTA TTG CTC AGC
T3 17 AAC CCT CAC TAA AGG GA
SK 17 TCT AGA ACT AGT GGA TC
KS 17 CGA GGT CGA CGG TAT CG
SP6 18 ATT TAG GTG ACA CTA TAG

Archiving of samples

For a while now the GGF has archived samples and primers from all single tube sequencing customers.  This free service has come at a great expense in technician time and freezer space.  With the new mission of the GGF as becoming a full service genomics facility, we no longer have the ability to maintain the permanent archives and will be phasing out this service as of August 2009.  For those of you that currently have archived samples, we will be contacting PIs to arrange their return.  If you do not wish them returned, please let us know and we will dispose of them.  For new samples coming in August and beyond, we will be keeping the samples and primers for two weeks to allow re-runs if necessary.  After two weeks, the samples and primers will be disposed of, unless prior arrangements were made to return them.  If you want the samples back please let us know and we will gladly return them.

For customers who do a large volume of samples with the same primers (e.g., diagnostics), we can archive those primers if requested.  For customers that are doing repeated sequencing with the same sample (e.g., primer-walking), special arrangements can be made to archive those samples for short-term experiments.

Ready-to-Run sequencing

For customers that want to do the cycle sequencing for themselves can submit reactions in single tubes or in 96-well plates.  Reactions should be submitted wet (if submitted dry, please let us know how much water to rehydrate them in) rehydrated in high quality molecular grade water.  The reactions have to be in semi-skirted barcoded plates that are compatible with 3730xl.  Compatible plates include: Applied Biosystems (#4306737), or AbGene (#AB-2400).  Where barcoded plates are not available, the GGF can supply barcodes as long as samples are submitted in semi-skirted plates.

Ready-to-Run genotyping

For customers that want to do fragment analysis.  Reactions should be submitted in deionized formamide with an internal lane standard included.  Similar to sequencing, the reactions have to be in semi-skirted barcoded plates that are compatible with 3730xl.  When submitting you must specify which fluorescent chemistry you are using.  As we have different protocols for 4-color (FAM, VIC, NED, ROX) and 5-color (FAM, VIC, NED, PET, LIZ).  Samples cannot be run until we know which chemistry was used.

Sample submission

Sample submission for sequencing and genotyping should be made through the Office of Research Services web portal

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