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)	1.6 μl	1.9 μl	1 µM
Reverse Primer (10 µM)	1.6 μl	1.9 μl	1 µM
2X Supergreen PCR Master Mix	7.8 μl	9.4 μl	-
ddH20	4.1 μl	4.9 μl	-
Total volume	15 μl	18 μl	-

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

Transfer 15µL 1' PCR master mixutre in 8-strip PCR tubes.

Add 0.6µLDNA template in 8-strip PCR tubes, resulting in a 15.6µL reaction mixture for 1' PCR.

Negative control contains only 15µL master mixture but not DNA template

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	95 ºC	30-180 (a)
Denaturation	98 ºC	15	20-25 cycles
Annealing	64-68 ºC varied (b)	15
Extension	72 ºC	60-180 (c)
Final extension	72 ºC	210
Preservation	Preservation	4 ºC	∞

a. Varied depend on template complexityb. Annealing varied, 62-68C is working; Refer to 1' PCR primers for annealing temperaturec. 1kb ~ 1min extension; enough time allow full extension of sequence

1' hear-primers used in Huang lab

A	B	C	D
Name	Sequence	Tm°C	CG%
NS1B1ngs_H1	GCTATGCGCGAGCTGCcctngttgatyctgccagt	71.7	60
ITS4ngs_H1	GCTATGCGCGAGCTGCtcctscgcttattgatatgc	69	55.6
LR5_H1	GCTATGCGCGAGCTGCtcctgagggaaacttcg	70.2	60.6
EF1-526F_H1	GCTATGCGCGAGCTGCgtcgtygtyatygghcaygt	71	59.3
EF1-1567R_H1	GCTATGCGCGAGCTGCachgtrccrataccaccratctt	70.6	56
EF1-2218R_H1	GCTATGCGCGAGCTGCatgacaccracrgcracrgtytg	72.2	60.3
Ben2f_H1	GCTATGCGCGAGCTGCtccagactggtcagtgtgtaa	70.5	56.8
Bt2b_H1	GCTATGCGCGAGCTGCaccctcagtgtagtgacccttggc	74.5	62.5
T22_H1	GCTATGCGCGAGCTGCtctggatgttgttgggaatcc	70.3	56.8
RPB2-3bF_H1	GCTATGCGCGAGCTGCggwggwtayttyatyatyaatgg	65.6	48.7
RPB2-7cR_H1	GCTATGCGCGAGCTGCcccatrgcttgyttrcccat	72.3	59.7
fRPB2-11aR_H1	GCTATGCGCGAGCTGCgcrtggatcttrtcrtcsacc	71.7	60.8

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.
2X Supergreen PCR Master Mix	10.75 µL	12.9 µL	-
ddH20	10.75 µL	12.9 µL	-
Total volume	21.5 µL	25.8 µL	-

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

Transfer 21.5µL of the 2' PCR master mixture to 8-strip PCR tubes.

Add 2.5µL pre-mixed barcoded-head primers (Forward + Reverse) to each PCR tubes.

Add 1µL of 1' PCR product as template , resulting in 25µL reaction mixture for 2' PCR.

Negative control contains only 24µL 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	10-15 cycles
Annealing	64-68 ºC varied (a)	15
Extension	72 ºC	60 (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