??DNA Sequencer Sample Requirements
?
| ??  |
??????????????General Requirements
Important: E-mail completed sample sheets to sequence@sun.ac.za
- Download the form in the downloads section.
- Sample names should not be longer than 25 characters. Only alpha-numeric characters are allowed as well as the following special characters # - _
- Please do not use spaces in your sample names.
- All primers and DNA samples must be of high quality. It should not contain EDTA, salt, ethanol, RNA or phenol.
- Rather give too much volume than too little.
- If you are unsure of your DNA or Primer concentrations ask us to verify it.
?Requirements for DNA Sequencing
- If the template is a PCR product, it has to be purified of all PCR components before it is handed in for sequencing (or a clean-up should be requested).
- Plasmid DNA (per reaction): 5?l DNA at a concentration of 100ng/?l per reaction.
- Primers for sequencing: 5?l per reaction at a concentration of 1.1pmol/?l
The table below is a good starting point for optimization of PCR product concentrations. Please note that certain browsers display microliter (?l) as milliliter (ml).
| PCR product size | Concentration required (min volume of 5?l per reaction) |
| < 500bp | 5ng/?l |
| 500 - 1000bp | 10ng/?l |
| > 1000bp | 20ng/?l |
Sequences of available primers (5’-3’):
| M?13F?? | GTT TTC CCA GTC ACG AC |
| M13R | CAG GAA ACA GCT ATG AC |
| T3 | ATT AAC CCT CAC TAA AGG GA |
| T7 promoter | TAA TAC GAC TCA CTA TAG GG |
| T7 terminator | GCT AGT TAT TGC TCA GCG G |
| SP6 | TAT TTA GGT GAC ACT ATA G |
| pGEX 5’ | GGG CTG GCA AGC CAC GTT TGG TG |
| pGEX 3’ | CCG GGA GCT GCA TGT GTC AGA GG |
| EGFP-N | CGT CGC CGT CCA GCT CGA CCA G |
| EGFP-C | CAT GGT CCT GCT GGA GTT CGT G |
Requirements for Fragment Analysis
The Sequencing facility also provides a fragment analysis service. Please contact the facility for more information BEFORE ordering primers so that we can ensure that the labeled primers are compatible with the instruments in the facility. We can also assist you in organizing your primer sets so that you can get as much data per run as possible. The costs below do NOT include amplification of the fragments. Results are returned in GeneMapper format. There is a version of PeakScanner and Microsat analysis that you use in the ThermoFisher cloud space that can analyse?.fsa files generated by the 3500xl instrument. The link for this software is https://www.thermofisher.com/za/en/home/order.html. You register on the website and then click on Connect: Lab, Data, Apps to access the software.
Requirements for DNA Extraction
Please supply all tissue in clearly labeled containers. It is always advisable to contact the facility in advance to ensure that we follow an extraction protocol that will yield the results you need.
??Requirements for PCR Setup
Please supply the DNA in a clearly marked container. If you are submitting a large number of samples it will help us a lot if you could submit the samples in a 96 well format if possible. It enables the use of multichannel pipettes which cuts down on handling time, which in turns enable us to process your sample faster. You also need to supply us with either the primer sequences or enough primer to process your samples. We can return any excess primer and DNA to you afterwards. Any information regarding the PCR conditions will be helpful.
Requirements for MLPA
Please supply the DNA/RNA in clearly marked tubes, after the MLPA reactions have been completed. If you are submitting a large number of samples it would help us a lot if you could submit the samples in strips of eight or 96 well format, if possible. Please supply us with details about the label that is used as well as specifications from suppliers, if available. Results are returned in GeneMapper format. There is a version of PeakScanner and Microsat analysis that you use in the ThermoFisher cloud space that can analyse.fsa files generated by the 3500xl instrument. The link for this software is https://www.thermofisher.com/za/en/home/order.html. You register on the website and then click on Connect: Lab, Data, Apps to access the software.
Requirements for SNaPshot Analysis
Please supply the DNA/RNA in clearly marked tubes, after the MLPA reactions have been completed. If you are submitting a large number of samples it would help us a lot if you could submit the samples in strips of eight or 96 well format, if possible. Please supply us with details about the label that is used as well as specifications from suppliers, if available. Results are returned in GeneMapper format. There is a version of PeakScanner and Microsat analysis that you use in the ThermoFisher cloud space that can analyse.fsa files generated by the 3500xl instrument. The link for this software is https://www.thermofisher.com/za/en/home/order.html. You register on the website and then click on Connect: Lab, Data, Apps to access the software.
?Requirements for Next Generation Samples
Please contact ngs@sun.ac.za if you’d like one of the NGS team members to get in touch regarding costs or study design for your NGS project.
Download sample preparation form here.?
?
