Capillary Sequencing The DNA Sequencing Shared Resource provides cost effective and rapid DNA sequence analysis using an ABI3730 instrument. The DNA Sequencing Facility offers full service fluorescent-based capillary DNA sequencing. Users can submit samples of PCR products, single and double stranded plasmids. Facility staff set up reactions, run the samples and post final data on a shared drive for retrieval. The resource also provides consulting expertise for DNA preparation to improve the DNA sequencing results of the users. The on-site service provides convenience and a rapid turn-around time, facilitating the many projects that require DNA sequencing support.
The DNA Sequencing Shared Resource provides full service DNA sequencing using fluorescence based technology, and access to high throughput next generation sequencing technologies.
For ABI3700XL sequencer, the optimum amount of DNA in the reaction depends on the nature of the template. The Manufacturer's recommended range for DNA quantity for specific templates is below.
TEMPLATE
SIZE
CONCENTRATION
PCR Product
100-500 bp
5 ng/µl
500-1000 bp
5 to 10 ng/µl
1000-2000 bp
10 to 20 ng/µl
>2000 bp
20+ ng/µl
Single-Stranded
20 to 50 ng/µl
Double-Stranded
100 to 125 ng/µ
Other template requirements:
DNA templates must be resuspended in Milli-Q H2O. Provide AT LEAST 5µL per reaction of template sample for each reaction. Quantitate purified templates with a spectrophotometer set at 260 nm or Nanodrop/Agilent BioAnalyzer:
o Single Stranded DNA: One OD unit equals 33 ng/µL. o Double Stranded DNA: One OD unit equals 50 ng/µL. o A260/A280 and A260/230 ratios should have a value above 1.8. Smaller ratios usually indicate contamination by proteins or organic chemicals.
PRIMERS
Primers must be resuspended in Milli-Q H2O and at least a concentration of 0.5 pmoles/µL and at least 10 µL supplied per reaction. You can submit your own primer with your sample, or you can request use of a facility primer, provided at no charge. The primers below are the ONLY ones that this facility orders. They are HLPC-purified and quantified to this facility's protocols.
M13 Forward Primer: 5' GTT TTC CCA GTC ACG AC 3' M13 Reverse Primer: 5' AAC AGC TAT GAC CAT G 3' T7 Primer: 5' GTA ATA CGA CTC ACT ATA GGG C 3' T3 Primer: 5' AAT TAA CCC TCA CTA AAG GG 3' EGFP-C Primer: 5' CAT GGT CCT GCT GGA GTT CGT G 3' EGFP-N Primer: 5' CGT CGC CGT CCA GCT CGA CCA G 3' MIR 30-5 Primer: 5' TGT TTG AAT GAG GCT TCA GTA C 3' MSCV-5 Primer: CCC TTG AAC CTC CTC GTT CGA CC MSCV-3 Primer: GAG ACG TGC TAC TTC CAT TTG TC
Please double-check the sequence to make sure that the primer sequence will work with your templates.
PROTOCOLS FOR 96 WELL PLATE SAMPLE SUBMISSIONS
48 or 96 DNA samples can be submitted for sequencing in a 96 well plate format. These will be sequenced as a single injection so it will only generate 1 text and 1 electropherogram file. These reaction products will be loaded entirely onto the sequencer, so there would be no aliquots to re-inject if a reaction fails. This protocol does not have overloaded sample issues. The cost, regardless of number of wells used, is $150 per plate.
To submit samples: - Send at least 5uL of template (approximately 200ng/uL for plasmids) per well in a 96 well half skirted, conical well, (NEVER round bottomed) reaction plate. - Send a tube containing at least 30uL of primer (@ 25.0 uM). We can only process this method with one or two primers per plate and if you are using two primers then the entire plate must be split in half (i.e., top half rows, A-D for one primer, rows E-H for the second primer). We do NOT stock any primers at this concentration. - Seal the plate, spin down & freeze. - Fill out the on-line submission for "96 Well Plate" submission. - Drop off, with the submission form, at one of the drop off locations on the main campus or Woodbury.
To retrieve data: The sequencer assigns a unique name to each file in the following format: “aae84aaa01aaa01aaa01.b1b1b2b3_A01_aae84a01.b1_015.ab1”
This is how your files (both the text and the electropherogram files) will be named and posted to the server. In this example, the plate name “aae84” (in red) is followed by the well position “a01” (in green). Note that your actual file names will not be color coded.
Data Access
The "Sequences" server space is designated for temporary storage and transfer of the sequence data generated by this facility. Please copy any data you wish to keep on a removable disk or CD. Data more than 90 days old will be purged automatically.
Currently, for every reaction requested four files are generated, two text files (".seq") and two electropherogram images (.ab1"). The electropherograms are included so users can determine the quality of their results and therefore the reliability of the basecalling data. Samples are injected twice, first with the normal water resuspension and secondly with the same suspension diluted in half with formamide (the second text and image files for the reaction, which include "-form" in their file names) to yield the optimum data for the reaction. The two injections migrate differently and the dilution resolves any overloading of the capillaries and late injection issues. Please check both injections for the data you need for your project.
48 to 96 DNA samples can be submitted for sequencing in a 96 well plate format. These will be sequenced as a single injection so it will only generate 1 text and 1 electropherogram file. These reaction products will be loaded entirely onto the sequencer, so there would be no aliquots to re-inject if a reaction fails. This protocol does not have overloaded sample issues. The cost, regardless of number of wells used, is $150 per plate for internal users and $175 for external users.
To submit samples: - You may leave one well empty (a01) for a facility control reaction. - Send at least 5uL of template (approximately 200ng/uL for plasmids) per well in a 96 well plate. - Send a tube containing at least 30uL of primer (@ 25.0 uM). We can only process this method with one or two primers per plate and if you are using two primers than the entire plate must be split in half (i.e., top half rows, A-D for one primer, rows E-H for the second primer). We do NOT stock any primers at this concentration. - Seal the plate, spin down & freeze. - Fill out the on-line submission for "96 Well Plate" submission. - Drop off, with the submission form, at one of the drop off locations on the main campus or Woodbury.
To retrieve data: The sequencer assigns a unique name to each file in the following format: “aae84aaa01aaa01aaa01.b1b1b2b3_A01_aae84a01.b1_015.ab1”
This is how your files (both the text and the electropherogram files) will be named and posted to the server. In this example, the plate name “aae84” (in red) is followed by the well position “a01” (in green). Note that your actual file names will not be color coded.
48 to 96 DNA samples can be submitted for sequencing in a 96 well plate format. These will be sequenced as a single injection so it will only generate 1 text and 1 electropherogram file. These reaction products will be loaded entirely onto the sequencer, so there would be no aliquots to re-inject if a reaction fails. This protocol does not have overloaded sample issues. The cost, regardless of number of wells used, is $150 per plate for internal users and $175 for external users.
To submit samples: - You may leave one well empty (a01) for a facility control reaction. - Send at least 5uL of template (approximately 200ng/uL for plasmids) per well in a 96 well plate. - Send a tube containing at least 30uL of primer (@ 25.0 uM). We can only process this method with one or two primers per plate and if you are using two primers than the entire plate must be split in half (i.e., top half rows, A-D for one primer, rows E-H for the second primer). We do NOT stock any primers at this concentration. - Seal the plate, spin down & freeze. - Fill out the on-line submission for "96 Well Plate" submission. - Drop off, with the submission form, at one of the drop off locations on the main campus or Woodbury.
To retrieve data: The sequencer assigns a unique name to each file in the following format: “aae84aaa01aaa01aaa01.b1b1b2b3_A01_aae84a01.b1_015.ab1”
This is how your files (both the text and the electropherogram files) will be named and posted to the server. In this example, the plate name “aae84” (in red) is followed by the well position “a01” (in green). Note that your actual file names will not be color coded.
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