Restriction ligation

Digest DNA using FastDigest NdeI and XhoI according to the manufacturer protocol:

2 µl of 10X buffer
1 µg of DNA
1 µl of NdeI and XhoI
Water up to 20 µl

Incubated at 37° C for 1-2 hours.

1 µl of alkaline fosthatase (1U/µl) is added to the digestion reaction of vector and incubated for 15 minutes at 37 °C.

After restriction, the fragments were separated on gel and extracted using Thermo Fisher Scientific GeneJET Gel Extraction Kit and subsequently ligated using T4 DNA ligase in a backbone to insert 3:1 molar ratio:

2 µl of 10x T4 DNA Ligase buffer
100 ng digested linear vector DNA
Insert digested DNA 1:3 ratio
1 µl T4 DNA Ligase 1 U
Water up to 20 µl
Incubate 30 minutes at 22 °C.

Use 5 μL of the mixture for transformation of 50 μL of chemically competent E. coli cells