BstEII NEB#R0162

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	To release the insert, the TOPO clones were then digested by BstEII and NcoI (NEB, Ipswich, MA). After gel purification, these inserts were ligated using T4 ligase (NEB, Ipswich, MA) into TN6-GFP previously cut with BstEII and NcoI.

Protocol tips
To release the insert, the TOPO clones were then digested by BstEII and NcoI (NEB, Ipswich, MA). After gel purification, these inserts were ligated using T4 ligase (NEB, Ipswich, MA) into TN6-GFP previously cut with BstEII and NcoI.

Publication protocol

To facilitate cloning of the primary envelopes into the proviral DNA, we selected the TN6-GFP proviral DNA expression vector, an NL4-3-based construct modified to contain a BstEII restriction site 15 nucleotides (nt) after the signal peptide of NL4-3 env and a NcoI site at the end of the envelope (for map see Fig. 1 [24]). Primary envelopes were amplified with the sense primer C6323+ as previously described [24] (ttgtgGGTCACCgtctattatgggg) and the antisense primer ASenvNcoI (ctgcatCCATGGtttattgtaaagctgcttc). The PCR amplification was performed in 50 µl of a solution containing 100–250 ng of purified vector encoding the envelopes, 20 pmol of each primer, 200 µM dNTPs, and 1X buffer containing 15 mM MgCl2, and 2.6 U of Taq DNA polymerase (Expand High Fidelity PCR System, Roche). The PCR parameters were 94°C for 2 min to achieve initial denaturation, followed by 30 cycles at 94°C for 30s, 58°C for 30s, 72°C for 3 min and a final elongation at 72°C for 30 min. The PCR products were analyzed on 1% agarose gels, purified using QIAquick kit (QIAGEN, Valencia, CA) and subcloned into the TOPO XL vector (Invitrogen, Carlsbad, CA). To release the insert, the TOPO clones were then digested by BstEII and NcoI (NEB, Ipswich, MA). After gel purification, these inserts were ligated using T4 ligase (NEB, Ipswich, MA) into TN6-GFP previously cut with BstEII and NcoI. Ligation was performed in 20 µl of a solution containing 50 mM Tris-HCl (pH7.5), 10 mM MgCl2, 10 mM dithiothreitol, 1 mM ATP, and 25 µg/ml bovine serum albumin, and 2,000 U of T4 DNA ligase (NEB). Use of approximately 3 inserts per 1 proviral vector yielded high levels of ligation. To further increase the ligation efficiency, temperatures were alternated between 16° and 37°C every 30 sec. Half of the ligation products (i.e., 10 µl of the ligation reaction) were used to transform MAX Efficiency Stbl2 competent cells (Invitrogen). The resulting TN6-GFP clones containing the primary envelopes were then amplified and purified using a QIAGEN plasmid mega kit. Sequences were confirmed by sequencing.

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