2-step PCR mixture and conditions (Barcoded-head primers for seqs pooling)

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 μl	1.2 μl	0.4 µM
Reverse Primer (10 µM)	1 μl	1.2 μl	0.4 µM
ZEJU PCR Master Mix	12.5 μl	15 μl	-
ddH20	9.25 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	95 ºC	180
Denaturation	95 ºC	30	20-35 cycles
Annealing	60-66 ºC varied (b)	30
Extension	72 ºC	180
Final extension	72 ºC	420
Preservation	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

1' hear-primers used in Huang lab

A	B	C	D
Name	Sequence	Tm°C	CG%
NS1B1ngs_H1	GCTATGCGCGAGCTGCcctngttgatyctgccagt	71.7	60
LR5_H1	GCTATGCGCGAGCTGCtcctgagggaaacttcg	70.2	60.6
EF1-526F_H1	GCTATGCGCGAGCTGCgtcgtygtyatygghcaygt	71	59.3
EF1-2218R_H1	GCTATGCGCGAGCTGCatgacaccracrgcracrgtytg	72.2	60.3

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	10-15 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