dHPLC



SUPAMAC has appointed Dr Bing Yu as a consultant in dHPLC-related work and he can provide you with information about this technology.

Practical Notes

Template quality
Template DNA isolation methods that utilise spin columns, chaotrophic salts, organic extraction procedures, or dirty preparations in which cellular debris or organic compounds have not been removed will damage the DNASep Prep HT cartridge.

  

Information
 
Organic extraction methods must be followed by chloroform/isoamyl back-extraction and subsequent ethanol precipitation and wash. Spin column extractions must be followed with an ethanol precipitation and wash. A high fidelity enzyme with the ability to do proofreading during amplification is recommended. Do not add BSA to the PCR reaction. The storage and 10 x reaction buffers must be fully compatible with the separation cartridge. SUPAMAC recommends AmpliTaqTM Gold (Applied Biosystems) or OptimaseTM Polymerase (Transgenomic).
  

Services
 
 
Sample considerations
It is recommended that a wild type sample is available as a negative control. This control should be mixed with unknown samples to induce heteroduplex formation. In the case of a homozygous polymorphic site, there will be no heteroduplexes present without mixing with a wild type fragment of the same amplified region. All samples should be denatured and slowly reannealed including the wild type fragment. We recommend that you denature the amplicon at 95°C for 5 minutes and then slow cool to 25°C over 45 minutes using a PCR cycler.
 

Links
 
   

Import sequence and fragment description
The NavigatorTM software will provide a melting curve, a melt profile, and a prediction of the temperature to begin screening for mutation detection from your DNA sequence. There are 3 ways a sequence can be imported into Navigator:
Enter by copy/paste from a web browser or word processor.
Loaded from an ASCII text file on a floppy disk. Select File, open DNA Sequence and navigate the software to the floppy drive. The software will then prompt you that the sequence will be saved with a .dna extension.
Drag from an open folder or the Windows Explorer and drop onto the Sequence box.


Melting profile
If the location of the variant is known, you can use the melting profile charts to choose a particular melt temperature to run. Your fragment should be approximately 75% helical at the location of the variant at that run temperature. If the location is unknown, a melting curve (temperature titration) must be run to examine the behaviour of the fragment under denaturing conditions. Usually, running the predicted Tm +/-2C will give a good analysis of this behaviour.
Contact SUPAMAC if you need extra support in design and melting profile analysis.
Shipment of DNA
Please label the tops and sides of your tubes sequentially. SUPAMAC requires a 10µl minimum aliquot of your unpurified PCR product. This should be supplied in PCR tubes in strips of 8 (Interpath Services Cat # 313100, 20% discount on tubes for customers of SUPAMAC) or in 96 well plates (Eppendorf twin.tec PCR plates, semi-skirted, Cat # 0030 128.591). Please place your order online or fill in our order form and send this with your DNA by express mail.


Results
Results are retrieved online by internal customers or at a SUPAMAC terminal (please phone 02 9515 8403 for a booking).
TERMS AND CONDITIONS OF SUPPLY
Cost per sample:
SU/RPAH:$6 External: $7
Prices do not include GST.
You may incur further charges for support in design and melting profile analysis.
DNA of low quality will result in sub-optimal results. Our runs are stringently controlled therefore sub-optimal results are the responsibility of the researcher alone and the samples will be charged. Should your samples contain products that damage the cartridge then you will be charged for its replacement.
Printing costs $1/page