Wear glove, clean up the working bench w. 1% bleach

Prepare 1' PCR master mixutre for head-primers ( prepare 1.2X of solutions for pipetting error if needed )

PCR mixture for head-primers for each reaction

A	B	C	D
Component	Volume	Volume (1.2X)	Final conc.
Forward Primer (10 µM)	0.5 μl	1.2 μl	0.2 µM
Reverse Primer (10 µM)	0.5 μl	1.2 μl	0.2 µM
PowerPol 2X PCR Master Mix	12.5 μl	15 μl	-
ddH20	10.25 μl	11.1 μl	-
Total volume	23.75 μl	28.5 μl	-

Mix the 1' PCR master mixture gently by pippeting. Quick spin the tube.

Transfer 23.75µL 1' PCR master mixutre in 8-strip PCR tubes.

Add 1.25µLDNA template in 8-strip PCR tubes, resulting in a 25µL reaction mixture for 1' PCR.

Mix the reaction mixture gently by tapping the tubes. Quick spin the tubes.

Carry out PCR using the following condition:

1' PCR condition for head-primers

A	B	C	D
Step	Temp	Sec	Cycle
Initial denaturation	98 ºC	45
Denaturation	98 ºC	10	25 cycles
Annealing	60-66 ºC varied (b)	30
Extension	72 ºC	150
Final extension	72 ºC	300
Preservation	4 ºC	∞

b. Annealing varied, 60-66C is working; Refer to 1' PCR primers for annealing temperaturec. 1kb ~ 1min extension; enough time allow full extension of sequence

Carry out electrophoresis for inspection of DNA products

Markdown wells and upload the pictures to the Lab Google drive

Prepare 2' PCR master mixutre for barcoded-primers ( prepare 1.2X of solutions for pipetting error if needed )

PCR mixture for barcoded-primers for each reaction (NO PRIMERs at this point!!)

A	B	C	D
Component	Volume	Volume (1.2X)	Final conc.
ZEJU PCR Master Mix	7.5 µL	9 µL	-
ddH20	5.55 µL	6.66 µL	-
Total volume	13.05 µL	15.66 µL	-

Mix the 2' PCR master mixture gently by pippeting. Quick spin the tube.

Transfer 13.05µL of the 2' PCR master mixture to 8-strip PCR tubes.

Add 1.2µL pre-mixed barcoded-head primers (Forward + Reverse) to each PCR tubes.

Add 0.75µL of 1' PCR product as template , resulting in 15µL reaction mixture for 2' PCR.

Negative control contains only 14.25µL master mixture and premixed barcoded-head primers but not DNA template

Mix gently by tapping the tubes. Quick spin the tubes.

Carry out 2' PCR using the following condition:

2' PCR condition for barcoded-head primers

A	B	C	D
Step	Temp	Sec	Cycle
Initial denaturation	98 ºC	30
Denaturation	98 ºC	15	12 cycles
Annealing	64-68 ºC varied (a)	15
Extension	72 ºC	20 (b)
Final extension	72 ºC	210
Preservation	Preservation	4 ºC	∞

a. Annealing varied, 65 C is working based on test on 220531; Refer 2' PCR primers for annealing temperatureb. 1kb ~ 1min extension; enough time allow full extension of sequence

Carry out electrophoresis for inspection of DNA products

Markdown wells and upload the pictures to the Lab Google drive