Cloning strategy.

<p>Entry clone (A) and expression vector (B) are mixed in one tube together with BsaI and ligase. Of the 4 possible ligation products, A to D, only the desired product, D, is stable, while all others are redigested with BsaI. Numbers 1 to 8 denote any nucleotide of choice, and numbers in italics denote the complementary nucleotides. FOI, DNA fragment of interest.</p

Generation of entry clones lacking internal BsaI sites for genes ycf4 and psbC.

<p>(A) Three PCR fragments were amplified from <i>Arabidopsis thaliana</i> genomic (chloroplast) DNA using primers yc1–6 (gene ycf4). B, BsaI recognition sequence. The fragments were cloned in on tube into entry cloning vector pECV, resulting in entry clone pE-YCF4. Structure of the fragments for the psbC gene (not shown) is similar except that the wildtype gene contains only one internal BsaI site. (B) Restriction digest (BsaI) of minipreps from 10 white colonies for constructs pE-YCF4 and pE-PSBC. As a control, the ycf4 and psbC ORFs were amplified from genomic DNA with only the two flanking primers, and the PCR products run undigested (u, expected size 583 nt for ycf4 and 1450 nt for psbC) and digested with BsaI (d, expected sizes for ycf4: 332, 113, 54, 14, 10, expected sizes for psbC: 947, 469, 14, 10).</p

General strategy for generation of entry clones lacking internal BsaI sites.

<p>For each gene of interest, two primers are designed to introduce two BsaI flanking sites (<i>pr1, pr2</i>), as well as one pair of primers for each internal site to eliminate (<i>pr3, pr4</i>). Column-purified PCR products pr1–3 and pr2–4 are mixed together with entry cloning vector (pECV) and BsaI enzyme in restriction-ligation buffer. The mix is digested for 10 minutes and heat inactivated. Ligase is then added and the mix is ligated for 10 minutes before transformation in <i>E.coli</i>. All white colonies contain the expected entry clone, with two flanking BsaI sites but no internal site. Horizontal arrows represent parts of the primers identical to the target sequence. Small vertical arrows indicate the location of the introduced mutation.</p

Subcloning of 2 and 3 inserts into an expression vector.

<p>(A–C) Maps of expression cloning vector pX-lacZ (A), and of entry clones and the resulting constructs for 2 or 3 insert cloning experiments (B, C respectively). The positions of the restriction sites for the enzymes XmaI and BsrGI are shown as black arrows, and the expected fragment sizes indicated. (D) Restriction digest (XmaI and BsrGI) of 12 minipreps for the two insert cloning (pX-GFP-H, lane 1 to 12) and the three insert cloning (pX-S-GFP-H, lane 13 to 24), and one miniprep of the vector (pX-lacZ, lane V).</p

Efficiency of cloning for two or three inserts.

<p>Restriction-ligations were performed from 5 to 30 minutes in Promega ligation buffer, and followed by digestion and heat inactivation. Cloning for two or three inserts was performed in duplicate.</p>*<p>The zero minute time point was performed without adding BsaI to the restriction-ligation mix.</p

Model for subcloning of a gene of interest into a library of compatible expressions vectors.

<p>(A) A gene of interest can be cloned as a single fragment in one entry construct (EC) or cloned as separate fragments in several entry constructs (ECs). BsaI sites 1 to 4: B1–B4. Example of three expression vectors consisting of a viral provector (V1), a TMV-based viral vector (V2) and a standard non-replicating expression vector (V3), and of the expression constructs obtained after cloning (E1–3). GOI, gene of interest; pol, TVCV RNA-dependent RNA polymerase; m, movement protein; SP, signal peptide; P, promoter. (B) Overview of the cloning strategy from entry construct to expression vector. Boxes flanking the LacZ fragment (blue) represent elements specific to the various expression vectors.</p

Subcloning of one insert into an expression vector.

<p>(A) Maps of entry clone pE-GFP, expression cloning vector pX-lacZ and expression construct pX-GFP. Black arrows show the position of the restriction sites for the enzymes BseRI and HindIII, and the numbers next to these indicate the sizes of the restriction fragments obtained. The grey triangle represents a <i>Streptomyces</i> phage C31 attB recombination site (this site is not used for the cloning procedure described here). Z, LacZ alpha fragment; N, Viral 3′ Non-translated region; T, Nos terminator; RB/LB, T-DNA right/left borders; S1–S2, selectable markers 1 and 2 (resistance to carbenicillin and kanamycin, respectively). (B) Plasmid DNA from 48 white colonies and vector digested by BseRI and HindIII and run on a 1% agarose gel. The upper and lower panels show minipreps obtained from cloning performed using ligation buffer or NEB buffer 3, respectively. DNA from all white colonies has the restriction pattern of pX-GFP (the 119 bp fragment is too faint to be visible on the picture). M: GeneRuler 1kb DNA Ladder Plus from Fermentas. V, vector pX-lacZ.</p

Efficiency of cloning of pX-GFP at different restriction-ligation times.

<p>Restriction/ligations were performed from 5 to 30 minutes, and either immediately transformed in <i>E.coli</i> (Digest an heat inactivation/no) or incubated 5 minutes at 50°C and 5 minutes at 80°C (Digest an heat inactivation/yes) before transformation in <i>E.coli</i>.</p>*<p>The zero minute time point was performed without adding BsaI to the restriction-ligation mix.</p

Test of QC cloning performed with or without heat inactivation.

<p>(<b>A</b>) PCR product amplified from G-tailed cDNA prepared from biopsy sample T019 using primers bap2 pc and GC3F. (<b>B</b>) Structure of the vector and of the PCR product. (<b>C</b>, <b>D</b>) The PCR product was cloned into pICH31480 using T4 DNA polymerase treatment for 5 minutes at 25°C (A, adaptor; U, unknown sequence; K, known sequence; CS, catching sequence), followed by heat inactivation 20 min at 75°C (<b>C</b>) or incubation at 4°C (<b>D</b>). Eight randomly chosen clones were analyzed by colony PCR using vector primers. The products amplified by colony PCR were separated on a 1% agarose gel supplemented with ethidium bromide and visualized under UV light. The expected insert size is indicated by an arrow.</p

Test of QC cloning using Klenow DNA polymerase.

<p>(A) Test of Klenow exonuclease activity determined using the same assay used for T4 DNA polymerase. (B) To test QC cloning using Klenow DNA polymerase, the PCR product T019 GC3F was cloned into pICH31477 (23 nucleotide catching sequence) and pICH31480 (52 nucleotide catching sequence). Incubation was performed at 37°C for 0, 30, 60, 90, and 120 minutes. (<b>C</b>) Eight randomly chosen clones from 120 min time points were analyzed by colony PCR using vector primers. The size of the expected full-length fragment is indicated by an arrow.</p