Journal: PLoS ONE
Article Title: A Mammalian Cell Based FACS-Panning Platform for the Selection of HIV-1 Envelopes for Vaccine Development
Figure Lengend Snippet: Schematic overview of the QL cloning procedure. An envelope gene or an envelope library is amplified with primers to introduce flanking Esp3I restriction sites enabling the generation of a 5′ NcoI and a 3′ Xho sitey (A; top). The envelope gene or an envelope library is incubated together with pQL9/11 in a one-tube reaction with Esp3I and T4-Ligase. Compatible “sticky-ends” (equally colored) can be ligated successfully, direct proper orientation and mediating resistance for further cleavage (A). Following transformation of CcdB sensitive bacteria, only recipients bearing a plasmid without CcdB are able to form colonies in the presence of ampicillin. (B) The lentiviral vector construct pQL9 comprises (i) 5′LTR (Long terminal repeat), (ii) EF1α (human promotor), (iii) GFP (marker gene), (iv) an IRES (internal ribosome entry site), (v) a CcdB positive selection marker  , and (vi) a 3′LTR sequence.
Article Snippet: Meanwhile a second reaction for the ligation was prepared. (II) 3 µL 10 mM ATP, 1 µL 10 x Tango Buffer, 1 µL 10 mM DTT, 1 µL T4-Ligase (NEB) addition of H2 0 to reach 10 µL.
Techniques: Clone Assay, Amplification, Introduce, Incubation, Transformation Assay, Plasmid Preparation, Construct, Marker, Selection, Sequencing