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The Massey Genome Service supplies the following primers for customers using the Full Sequencing Service:
Customers can request to use these primers by ticking the appropriate box or boxes on the online sequencing request form in the section called MGS primers and by indicating in the column called Primer Name which primer to use with each template.
The Massey Genome Service primer sequences are:
• |
M13 forward: |
5' CCC AGT CAC GAC GTT GTA AAA CG 3' |
• |
M13 reverse: |
5' AGC GGA TAA CAA TTT CAC ACA GG 3' |
• |
T7 (Promoter/forward): |
5' TAA TAC GAC TCA CTA TAG GG 3' |
• |
SP6 (Promoter): |
5' ATT TAG GTG ACA CTA TAG 3' |
The Massey Genome Service uses the following sequencing chemistries for the Full Sequencing Service:
Although all of the chemistries are relatively versatile, some are better than others for specific types of templates. The Massey Genome Service uses the BigDye™Terminator Version 3.1 chemistry for most sequencing unless the customer specifies the use of one of the other two chemistries. Please indicate which type of sequencing chemistry you want the Massey Genome Service to use if requesting the Full Sequencing Service, by ticking the appropriate box on your online request form.
Below is a table showing the sequencing kits the Massey Genome Service uses, and the types of sequencing applications work best with each chemistry:
Applications |
BigDye® TerminatorV3.1 Chemistry |
|
dGTP BigDye® TerminatorV3.0 Chemistry |
De novo sequencing |
R |
R |
|
Re-sequencing |
R |
R |
|
Sequencing difficult Templates |
R |
R |
|
Long-read sequencing |
R |
R |
|
Sequencing across all template types (plasmids, PCR products, BACs, cosmids, and bacterial genomic DNA) |
R |
R |
|
Mixed-base detection |
R |
S |
|
Sequencing short PCR Products using rapid Electrophoresis run modules |
S |
S |
R= Recommended S= Satisfactory
BigDye® Terminator V3.1 Chemistry: This chemistry is designed for the majority of applications, and produces data with uniform peak heights and optimized signal balance to produce long reads. Improved peak patterns also contribute to more accurate base assignments for heterozygote and mutation detection.
dGTP BigDye® Terminator V3.0 Chemistry: This chemistry is designed to sequence through GT and GA repeats, and G homopolymers that the BigDye® Terminator V 3.1 chemistry has not been able to get through. But the BigDye® Terminator V 3.1 chemistry will generally sequence through most repeats.
The Massey Genome Service requires customers who are using the Full Sequencing Service to send the template and primers premixed in 0.2ml individual PCR tubes or 0.2ml strip tubes. For the Sequencing Capillary Separation Service and Sequencing Capillary Separation Service with reaction cleanup please send the sequencing reactions in 0.2ml individual PCR tubes or 0.2ml strip tubes and follow the guideline below for setting up your sequencing reactions. For the Sequencing Plate Service and Sequencing Plate Service with reaction cleanup, please send the sequencing reactions in a 96 well plate, sealed with strip tubes, plastic seal or foil seal. The strip tubes provide the best result.
Template Type |
Template Total Quantity(in final volume) |
Primer Total Quantity*(in final volume) |
Final volume required # |
PCR product: Rule: For PCR products use 2.5ng of template for every 100bp. |
|
|
20ul if using your own primer 19ul if using MGS primer |
Single-stranded Plasmid |
62.5-125ng |
4pmol |
20ul if using your own primer 19ul if using MGS primer |
Double-stranded Plasmid |
250-625ng |
4pmol |
20ul if using your own primer 19ul if using MGS primer |
Cosmid, BAC DNA, and Lambda DNA |
0.625-1.25ug |
6.4pmol |
20ul if using your own primer 18ul if using MGS primer |
Bacterial Genomic DNA |
2.5-3.75ug |
6.4pmol |
20ul if using your own primer 18ul if using MGS primer |
* Make sure only one primer is added to the template/primer premix
# Make the template/primer premix up to the final volume with filtered molecular grade water.
Example:
You have a 500bp PCR product at a concentration of 6.25 ng/µL and your primer is at a concentration of 2 pmol/µL. For PCR products use 2.5ng of template for every 100bp. So for a 500bp product you will need to add 12.5ng to the template/primer premix. At a concentration of 6.25 ng/ µL you will need to add 2 µL template to the premix. For the primer you need 4pmol total amount in the template/primer premix. So at a concentration of 2 pmol/µL you will need to add 2 µL primer to the premix. You then need to make the premix up to a final volume of 20 µL. So you will need to add 16 µL of filtered molecular grade water to the premix to get the final volume of 20 µL.
Poor template quality is the most common cause of sequencing problems. The following are characteristics of poor quality templates:
The quality of DNA in a reaction can affect the performance of the ABI3730 DNA Analyzer. Potential contaminants include:
The presence of residual salts, proteins, RNA and detergents can interfere with capillary electrophoresis and electrokenetic injection. Capillary electrophoresis is especially susceptible to salt in samples, either from template preparation, from cycle sequencing reactions, or from precipitation methods using salts. The negative ions in salts can be preferentially injected into the capillary array during electrokenetic injection, leading to low signal. In addition, the negative ions compete and interfere with the injection of lager DNA extension fragments, leading to shortened read lengths.
Please QC all DNA templates; PCR products/plasmids, cosmids, BACs or bacterial genomic DNA, by spectrophotometry or fluorometry. The OD260/280 for DNA should be 1.8 to – 2.0.
If you think any of your samples have either a high GC content and/or secondary structures, please request in the "Customer Notes" section on your online request form, to have 5% DMSO added to your samples.
If you suspect your templates have difficult to sequence repeat and homopolymer regions, please select in the "Reaction Type" section on your online request form, the dGTP BigDye Terminator v 3.0 chemistry. However, please note that if the repeat or homopolymer regions in your templates are long and complex, the dGTP BigDye Terminator v 3.0 chemistry still may have difficulty sequencing through this difficult region.
If you have either cosmids, BACs or bacterial genomic DNA, please select in the "Procedures" section on your online request form, full sequencing service for BAC templates or bacterial genomic DNA. This is required to run your samples using the correct protocol.
Please note, the "Capillary Separation" service is a more cost effective option for sequencing. If you would like more information please don't hesitate to contact us.
The following methods can be used to examine DNA quality:
Agarose gels reveal the presence of contaminating DNAs & RNAs, but not proteins. Spectrophotometry can indicate the presence of protein contamination, but not DNA & RNA contamination. These two methods should be used together to get the most information about your DNA template before sequencing.
Neither of these methods shows the presence of contaminating salts that can cause noisy data. If you suspect that your DNA is contaminated with salt, remove the salt before sequencing. The most efficient method for salt removal is ultrafiltration with a Centricon-100 column. Spin columns & ethanol precipitation can also be used.
Primer Design Recommendations
The following recommendations are provided to help optimize primer selection:
Download the attached Sequencing Technical Bulletin below, which contains information on the following:
MGS ABI Sequencing Service Technical Bulletin - February 2019.pdf (485 KB)
Download the attached ‘QualTrace™ Bulletin’ below, which contains information on the following:
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Last updated on Thursday 11 February 2021
Contact us Mon - Fri 8:30am to 4:30pm 0800 MASSEY (+64 6 350 5701) TXT 5222 contact@massey.ac.nz Web chat Staff Alumni News Māori @ Massey