Amplifying the target genomic region by PCR

For a 150bp paired-end or single-read sequencing experiment, design primers in a way that the region of interest, like CRISPR cutting site or specific mutations, locates within 130bp from at least one of the primers

Add NGS adapters to 5’ end of primers following the instructions of your local NGS facility

20 µl PCR reaction for each sample

PCR Reaction - setup

A	B
Ultrapure H2O	10.6 µl
5x HF buffer	4 µl
2.5 mM dNTP	1.6 µl
10 µM primer Forward	0.5 µl
10 µM primer Reverse	0.5 µl
DMSO	0.6 µl
Titan DNA polymerase or Phusion	0.2 µl
Crude cell lysate	2 µl

Calculate the amount of each component needed for all samples, also count a negative control and positive control

In a pre-PCR area, mix all components except the crude cell lysate to a microcentrifuge tube or a 15 ml conical tube to make master mix

Aliquot 18 µl to each 200 µl microcentrifuge tube or each well in a 96-well PCR plate. Use reservoir and multi-channel pipet if there are many samples.

Add 2 µl crude cell lysate to the reaction. In one reaction, use 2 µl H2O instead as negative control. In another reaction, use 2 µl previously validated crude cell lysate as positive control.

Cap the tubes or seal the plates properly with an adhesive seal.

Shake the tubes or plates vigorously to mix

Briefly spin the samples

Use the following touch-down PCR protocol in a thermocycler

Touch-down PCR protocol

A	B
98°C	3min
98°C	30s
70°C (touch down, 1°C/cycle)	30s
72°C	30s
Go to 2	12 cycles in total
98°C	30s
58°C	30s
72°C	30s
Go to 6	23 cycles in total
72°C	7min
4°C or 12°C	forever