What are the components of the IHG kit?

Forward and reverse primers for both genomic DNA and IHG, the IHG, control DNA for all known alleles, instruction sheets.

How many reactions are in a kit?

10, 25 or 50 as standard, but other requests can be accommodated.

Do you need to extract genomic DNA before the PCR reaction or can raw blood/tissue be added to the tube?

We recommend extracted DNA but crude lysates of blood, buffy coat or any nucleated cell preparation should produce a result.

If the DNA is extracted do you need to quantify it in order to know how much needs to be added to the PCR reaction to achieve enough amplification to analyse?

We recommend 100ng-500ng per reaction but there is considerable flexibility. Concentrations between 10ng and 1ug per reaction have been successfully used in over 24,000 clinical samples.

Can multiplexed IHG be run to investigate several SNPs or diseases at the same time?

IHG can be multiplexed, depending on what tests are required. For example, FVL, PTR and MTHFR is a 3-SNP multiplex. Combinations of different SNPs and different fluorochrome-labelled IHGs can be accommodated. Enquire for details.

How long does a test take, start to finish?

Assuming DNA is isolated and amplified, it takes about 15 minutes for the crossmatch, plus separation time. For capillary electrophoresis (e.g. on an Applied Biosystems 310, 3100 or 3130 instrument, allow 15 minutes per sample. For minigel PAGE this is 60-90 minutes and 5 minutes for the stain, a total approximately 80-110 minutes.

How many PCR cycles are required? Is the PCR reaction a standard, universal one which applies to all the kits?

The standard recommended is 30-32 cycles. This depends completely on the thermal cycler used, and can take 30 minutes (light cycler) to 3 hours. PCR conditions will vary from kit to kit but we try to accommodate most in a single program.

I wish to use minigel PAGE. What percentage gel is required for analysis? What size gel is required? How long will separation take and at what voltage?

We recommend 15% nondenaturing PAGE. Any size gel can be used but separation can be achieved in 60-90 minutes on a 7cm height gel or above. On a 7cm gel, we would use 200V (for a 10cm gel width) or 300V (for a 25cm gel width).