1. Because of the high sensitivity of this protocol, it is recommended that the outer PCR run in a single tube format and followed by either single tube or strip format for inner PCR. This is to reduce the possible cross contamination from one tube to the other.

2. The following solutions need to be prepared:

Tag 5U/µl
Primers
	TH132/TH133 (25 µM )	Outer PCR
	Mhp3/Mhp4 (5.5 µM )	Inner PCR
dNTPs	2 µM	Inner PCR
MgCl2	50 mM	Inner PCR
MasterAmp E	2X	Outer PCR
Qiagen buffer	10X	Inner PCR
Q solution	5X	Inner PCR
SuperQ H2O

Outer PCR:

3.1 There are 29 wells available for running 0.5 ml tubes at one time in a Ericopm TwinBlock thermocycler. Each run should includes the following controls:

Negative control: No DNA

Positive control: 100 fg/10µl

10 fg/10µl

2.5 fg/10µl (or 1 fg/10µl)

1 ng/10µl (or 0.5 fg/10µl)

3.2 This leaves 24 wells for clinical samples. If cross contamination is suspected to be a problem, it is recommend that more negative controls can be every other 5 clinical samples.

25 µM TH132	25 µM TH133	MasterAmp E 2X Buffer	H2O	5 U/µl Taq	Template DNA	Total volume
2 µl	2 µl	25 µl	11 µl	0.25 µl	10 µl	50 µl

3.3 Master solution should be prepared by mixing all the components except template DNA. Then, 40 µl is dispensed into each tube. The volume of the master solution should be prepared such that it is enough for extra one or two reactions than the number of samples intended to run.

3.4 The reaction includes 4 minutes denaturation at 94°C followed by 30 repeats of denaturation at 94°C for 1 minute, annealing at 48.8°C for 1 minute, and extention at 72°C for 1 minute. The reaction is then incubated at 72°C for 7 minute.

Inner PCR:

4.1 10 µl of outer PCR product is diluted into 990 µl H2O in eppendorf tube. The dilution is mixed by vortex followed by a short spin in a microfuge to make certain that no diluent was attached to the inner ring of the lid of the eppendorf tube, which might result in an increase of cross contamination during opening and closing of the eppendorf tube.

5.5 µM Mhp3	5.5 µM Mhp4	10X Qiagen Buffer	2 mM dNTPs	H2O	5X Q Solution	5U/ µl Tag	Template DNA	Total Volume
1 µl	1 µl	5 µl	1 µl	30 µl	10 µl	0.25 µl	2 µl	50 µl

4.2 Master solution should be prepared by mixing all the components except template DNA. Then, 48 µl is dispensed into each tube. The volume of the master solution should be prepared such that it is enough for extra one or two reactions than the number of samples intended to run.

4.3 The reaction includes 2 minutes denaturation at 92°C followed by 30 repeats of denaturation at 92°C for 1 minute, annealing at 56°C for 1 minute, and extention at 72°C for 1 minute. The reaction is then incubated at 72°C for 7 minute prior to analysis in 1 % agarose gel with 10 µl of reaction mixture.