Passage and harvest the cells following the cell culture protocol.

Pellet the cells, aspirate the supernatant, and transfer the pellet to a 1.5mL Eppendorf tube.

Add 1mL PBS and centrifuge at 400x g,0h 0m 0s for 0h 4m 0s.

Remove the supernatant very carefully.

Snap freeze the pellet and store it at -80°C for later genomic DNA extraction.

Add 50µL quick extract (QE) reagent to the pellet and vortex well. Aliquot QE reagent in 1.5mL tubes and store at -20°C.

Heat the tube of cell pellet in QE at 65°C for 0h 6m 0s.

Next, heat at 98°C for 0h 2m 0s.

Vortex well to dissolve the pellet completely.

Measure the DNA concentration using a nanodrop.

Set all reactions from here to the end with a positive control alongside the gDNA prepared above.

Incubate at 37°C in an incubator or heat block 0h 2m 0s.

Heat inactivate the reaction at 65°C for 0h 20m 0s.

Purify Sau3AI digested gDNA using PCR purification kit following manufacturer protocol. Elute DNA in 30µL H₂O.

Store the tube at -20°C for later.

Incubate the ligation reaction at 4°C (12 to 16 hours).

Heat inactivate the reaction at 65°C for 0h 20m 0s.

Cool down the sample to 65Room temperature.

Incubate at 37°C in an incubator or heat block 0h 20m 0s.

Purify EcoRV digestion reaction using PCR purification kit following manufacturer protocol. Elute DNA in 32µL TE buffer.

Store the tube at -20°C for later.

Set up the primary PCR amplification in a PCR tube.

A	B
Taq 2X Master Mix	10 µL
Spkt-Primer Mix-1	1.5 µL
Template (from step 6.5.3 elute)	5 µL
DNase/RNase-free H2O	3.5 µL
Final volume	20 µL

Run the reaction in the thermal cycler with the following program for Taq 2X master mix polymerase.

A	B	C	D
Initial denaturation	95°C	3 min	1X
Denaturation	95°C	30 sec	34X
Annealing	55°C	30 sec
Extension (1min/kb)	68°C	2 min
Final extension	68°C	5 min	1X
Hold	4°C	Forever

Store PCR product at -20°C.

Set up the secondary nested PCR in a PCR tube.

A	B
Taq 2X Master Mix	10 µL
Spkt-Primer Mix-2	1.5 µL
Template (from step 6.6.3)	1 µL
DNase/RNase-free H2O	7.5 µL
Final volume	20 µL

Run the reaction in the thermal cycler with the following program for Taq 2X master mix polymerase.

Store PCR product at -20°C.

Run the PCR product from step 28 on 1.5% agarose gel.

Gel purify each band separately. Store at -20°C.

Set up ligation reaction in PCR tube as following:

A	B
Gel-purified PCR product (from step 6.8.2)	0.8 µL (0.4-1.6 µL)
Salt solution	0.4 µL
Topo vector	0.4 µL
DNase/RNase-free H2O	0.8 µL
Final volume	2.4 µL

Incubate the reaction at -20Room temperature for less than 0h 30m 0s.

Transfer to ice or store at -20°C for later use.

Mix gently 1µL ligation reaction with 20µL competent cells in 1.5mL tube On ice. Incubate -20On ice for 0h 30m 0s.

Heat shock at 42°C for 0h 0m 45s. Leave 42On ice for 0h 2m 0s-0h 5m 0s.

Add 200µL 42Room temperature SOC medium to the transformed cells. Incubate at 37°C in an orbit shaker for 1h 0m 0s.

Plate 20µL and 100µL on pre-warmed agar plate supplemented with 100µg/ml ampicillin. Incubate at 37°C 1h 0m 0s.

Pick a few colonies from each plate. Inoculate into 5mL LB broth supplemented with 100µg/ml ampicillin. Incubate at 37°C in an orbit shaker 1h 0m 0s.

Extract the DNA using Qiagen DNA miniprep.

Provide 10µL volume of plasmid prep from step 39 in PCR tube. The final concentration needs to be 80ng/µl-100ng/µl.

Fill the submission form on vendor’s website. Choose their relevant in-house primer from their primer list and mention on the form.

Blast the genomic sequence on NCBI website for the exact locus the piggybac construct has integrated.