The Division of Human Genetics Genome Platform offers the following services:
1. DNA sequencing using the ABI 3100 Genetic Analyzer, which is capable of sequencing 16 samples per hour by capillary electrophoresis.
Completed purified sequencing reactions (BigDye Terminator ver3.1 cycle sequencing kit, Applied Biosystems) should be cleaned up (see protocols) without the pellet being reconstituted, and delivered to the Division of Human Genetics in the IIDMM by 10am or 3pm to be included in the daily runs. All samples must be accompanied with a completed request form. Collections can be arranged by request.
Please provide an email address so that the results can be mailed directly to you.
|PRICING||UCT user||non-UCT user|
|Sequencing reaction (Inclusive)||R100.00||R130.00|
|Clean-up & electrophoresis||R30.00||R50.00|
These rates are per sample. Special rates can be negotiated for large numbers of samples.
Please note the following.
- The quality of the DNA template is crucial for accurate sequencing. It is directly proportional to the quality of the template DNA. PCR products should be cleaned up prior to sequencing using either spin-columns or Qiaex II resin (recommended) on the eluate from gel slices.
- Plasmids should be purified using commercial kits (Nucleobond, Wizard, Qiagen).
- Accurate quantitation* is essential for successful sequencing reactions.
- The minimum concentration for dsDNA is 30ng/ul (optimal 5ul @100ng/ul).
- For PCR fragments the amount of DNA used per sequencing reaction is usually 3ng per 100bp. Do not exceed 60ngs or go below 1ng. [*ng/ul = 9 X fragment size (kb)]
Use 1ul primer @ 10 pmol/uL per sequencing reaction (20ul).
- Primers should be at 18 bases long.
- Avoid runs of an identical nucleotide especially Gs.
- Keep G-C content within 30-80% range.
- Primers with Tm >450C produce better results than those with lower Tm.
- For primers with G-C content < 50% it may be necessary to extend the primer sequence beyond 18 bases to keep Tm>450C.
- Use of primers >18 bases minimises the chance of a secondary hybridisation site on the target DNA.
- Avoid primers with secondary structure or those that can hybridise to form dimers.
*Too much DNA blocks the capillaries and gives poor results and may damage the equipment. Too little DNA will also give sub-optimal results.
**TE is not suitable as an elution buffer – substitute with sterile distilled water. Samples and primers should also be free of residual salts, protein, PCR primers, RNA, EDTA etc.
Recommended G-50 post cycle sequencing reaction clean up:
- Sephadex G-50 Fine cat: 17-0042-01 (Amersham-BioSciences)
- Clean commercial spin columns (re-use by boiling in distilled water)
- 0.0625grm G-50 per 1ml distilled water.
- Add sephadex to water and mix gently.
- Stand at room temp for at least 2.5 hours or preferably overnight.
- Autoclave in batches!
- Add magnetic stirrer bar and mix for 10mins at medium speed.
- While mixing add 600ul to spin column placed in a collection tube.
- Spin in microfuge (eppendorf, radius 73mm) at 3500rpm for 2 mins. Repeat.
- Tap tube to ensure that the sephadex is not touching the sides of the column
- Add 10ul (20ul) of sequencing reaction carefully onto the centre of the sephadex.
- Spin in microfuge (eppendorf, radius 73mm) at 3500rpm for 2 mins.
2. DNA genotyping using the ABI 3100 Genetic Analyzer which is capable of handling 2 X 96 well plates in a single overnight run. Samples in batches of 16 can be run during the day by as required. Please contact us so that we can assure the quickest possible turn-around time.
|PRICING||UCT user||non-UCT user|
|Per 16 samples||R320.00||R480.00|
|Per 96 samples||R1 440.00||R1 920.00|
3. The WAVE mutation detection screening system which utilises DHPLC (denaturing high performance liquid chromatography
Note, amplicons need to be evaluated prior to running on the Wave instrument in order to ascertain how many methods are required to optimally screen each product. Please contact the Facility for more information, before sending samples.
Dr Yolandi Roodt
Tel: +27 021 4066297 firstname.lastname@example.org
Ms Alina Esterhuizen
Tel: +27 021 4066456 email@example.com