Figure 2: Cloning of pGL3-U6-sgRNA-PGK-mRFP-T2A-PuroR. (a) The mRFP-T2A insert was amplified from MCS1-EF1α-RFP-T2A-Puro-pA-MCS2 via a touchdown PCR and analyzed by agarose (2%) gel electrophoresis (lane 1). A mock control (without the DNA polymerase) was conducted in parallel (lane 2). (b) The methylated pGL3-U6-sgRNA-PGK-puromycin vector DNA was linearized via PCR, and subject to agarose (0.7%) gel electrophoresis before and after DPN1 digestion (lanes 1 and 3 resp.). The mock control for PCR lacked DNA polymerase (lane 2). (c) Colony PCR and subsequent agarose (2%) gel electrophoresis were performed to identify clones with the mRFP-T2A insert. Clone #2 was selected for DNA sequencing analysis.