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[DESCRIPTION]
This is a protocol for <em>in vitro</em> transposon mutagenesis for introduction of internal epitope tags. It is from:<br />Zordan RE, Beliveau BJ, Trow JA, Craig NL, and Cormack BP (2015) <a href="http://www.genetics.org/content/200/1/47.long" target="_blank">Avoiding the Ends: Internal Epitope Tagging of Proteins Using Transposon Tn7</a>. <em>Genetics</em>  200:47-58; doi:10.1534/genetics.114.169482<br /><br />Please see the <a href="http://www.genetics.org/content/200/1/47.long" target="_blank">full manuscript</a> for additional details.<br /><br />This protocol delineates the steps to perform a small (20μl) “‐ enzyme” negative control reaction and a large (80μl) “+ enzyme” experimental mutagenesis reaction in parallel. The enzymes and buffers are mixed together in a master mix (5.5‐reaction sized, to account for pipetting error) and later split into appropriate reaction sizes.

[BEFORE_START]
We highly recommend performing diagnostic restriction digests on plasmid pools from each phase of this protocol. See the guidelines for details.

[GUIDELINES]
<strong>REQUIRED REAGENTS </strong><br />Purified TnsA, TnsB, and TnsC<sup>A255V</sup> enzymes <sup>3, 4</sup> <br />TnsA stock = 150ng/μl in Storage Buffer A<br />TnsB stock = 200ng/μl in Storage Buffer B <br />TnsC A255V stock = 500ng/μl in Storage buffer C <br />Tn7 donor vector: pRZ101 (Tn7‐FLAG donor vector). Dilute to 25ng/μl in 10mM Tris pH8.0 <br />Entry vector of target gene: DCW1 entry vector. Dilute to 50ng/μl in 10mM Tris pH8.0 <br />Invitrogen MegaX DH10B T1R electrocompetent E. coli cells <br />300mM Magnesium Acetate (MgOAc) <br />phenol:chloroform:IAA (25:24:1) (Amresco 0883‐100ml) <br />Chloroform <br />3M Sodium Acetate (NaOAc) <br />100% ethanol (EtOH) <br />70% EtOH <br />FseI (New England Biolabs R0588S) <br />PmeI (New England Biolabs R0560S) <br />ApaLI (New England Biolabs R0507S) <br />T4 DNA ligase and buffer  (New England Biolabs M0202S) <br />Gateway LR clonase II (Life Techonologies 11791‐100) <br />Qiagen Hi‐Speed MidiPrep kit (Qiagen 12643) <br />MegaX DH10B T1R Electrocomptent Cells (Life Technologies C6400‐03) <br />LB media <br />Oxoid Isosensitest media (Iso) (agar: OXCM0471B, liquid broth: OXCM0473B) <br />Carbenicillin (100mg/ml  = 1000x stock) <br />Kanamycin (30mg/ml = 1000x stock) <br />Trimethoprim (5mg/ml in DMSO = 500x stock) <br />ddH2O = doubly distilled (MilliQ) H2O<br /><br /><strong>BUFFERS</strong> <br />Use sterile‐filtered MilliQ water to make all buffers. If possible, make all buffers in plastic containers; residual detergent on glassware may inhibit the transposition reaction. <br /><br /><strong>Standard buffers </strong><br />20mM ATP in 125mM Tris pH 7.5 (store at ‐20 °C for at most 1 month) <br />20mM DTT in 125mM Tris pH 7.5 (store at ‐20 °C for at most 1 month) <br />100mM ATP in 250mM HEPES (pH8.0) – for making TnsC storage buffer <br />1M DTT in 150mM HEPES (pH8.0) – for making TnsA and TnsC storage buffers <br />1M DTT in 1M Tris pH 7.5 – for making TnsB storage buffer <br />10mM Tris  ‐ for elution of mutagenized plasmid pools from midiprep kit <br />50% glycerol <br /><br /><strong>Tns storage buffers </strong><br />Store at ‐20 <span class="st">°</span>C for at most 6 months. We do not recommend refreezing and rethawing of the storage buffers, so store them in small aliquots and discard after use. <br /><br /><strong>Storage buffer A </strong><br />A 25mM HEPES pH8.0 <br />150mM NaCl <br />1mM EDTA <br />1mM DTT (in HEPES) <br />10% glycerol    <br /><br /><strong>Storage buffer B </strong><br />25mM TrisHCl pH8.0 <br />500mM NaCl <br />1mM EDTA <br />1mM DTT (in Tris) <br />25% glycerol <br /><br /><strong>Storage buffer C </strong><br />25mM HEPES pH8.0 <br />1M NaCl <br />0.1mM EDTA <br />2.5mM DTT (in HEPES) <br />1mM ATP   <br />10mM MgCl2 <br />10% glycerol<br /><br /><strong>Diagnostic digests </strong><br />We highly recommend performing diagnostic restriction digests on plasmid pools from each phase of this protocol. This helps to characterize the proportion of each pool that is mutagenized, identify any lingering donor plasmid that persists in the expression pools, and helps determine the complexity of the pool (ie, is the Tn inserted at many locations throughout the target ORF?).   <br /><br />We suggest performing many restriction digests in parallel. Use an enzyme that cuts only in the donor backbone, one that cuts only in the expression backbone, one that cuts in the Tn7L, and one that cuts in Tn7R. Perform “no enzyme” controls, as well. Include one double digestion which cuts in the epitope tag and one in the backbone – this will determine the complexity of the mutagenized pool. Perform restriction digest on the following DNA samples: <br />1) Unmutagenized target donor vector <br />2) wt expression vector <br />3) mutagenized donor pool <br />4) xpC pool <br />5) xpT pool <br />6) xpT‐L pool <br />7) xpT‐L‐R pool<br /> <br /><strong>References</strong> <br />1. Brachmann, C.B. et al. Designer deletion strains derived from Saccharomyces cerevisiae S288C: a useful set of strains and plasmids for PCR‐mediated gene disruption and other applications. Yeast 14, 115‐132 (1998). <br />2. Winzeler, E.A. et al. Functional characterization of the S. cerevisiae genome by gene deletion and parallel analysis. Science 285, 901‐906 (1999). <br />3. Gamas, P. &amp; Craig, N.L. Purification and characterization of TnsC, a Tn7 transposition protein that binds ATP and DNA. Nucleic acids research 20, 2525‐2532 (1992). <br />4. Choi, K.Y., Li, Y., Sarnovsky, R. &amp; Craig, N.L. Direct interaction between the TnsA and TnsB subunits controls the heteromeric Tn7 transposase. Proceedings of the National Academy of Sciences of the United States of America 110, E2038‐2045 (2013).<br /><br /><br />

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