Gel clean up reaction

The mature polypeptide of human ACE2 (GenBank NM_021804.3) was cloned in to the XbaI-BamHI site of pLenti-CMV-MCS-GFP-SV-puro (Addgene #73582). IRES linker was previously inserted into pcDNA3-sACE2v4-sfGFP via BamHI restriction enzyme site. The pcDNA3-sACE2v4-IRES-sfGFP insert was removed by restriction enzymes using NheI- XhoI. Next, the NheI-XhoI sites were converted into XbaI-BamHI by PCR (Primers: Forward-TAGCCTAGAGCCACCATGTCAAGCT, Reverse-CACCTGATCCCATTTGTAG AGCTCATCCATGCCATG) to be compatible with pLenti-CMV-MCS-SV40-PURO vector. Amplicon size was evaluated through gel electrophoresis in agarose 1.2%. DNA band was excised from the gel and purified using gel clean up reaction (Macherey Nagel). DNA insert and linear destination vector were mixed at a 1:1 ratio respectively. T4 was used for the ligase reaction and incubated overnight at 16 °C. Successful ligation of the inserted vector was verified through gel electrophoresis in agarose 2%. The DNA band was excised from the gel and purified using gel clean up reaction (Macherey Nagel).