Editing Purification of Benzonase, SM2 Nuclease (section)

== Cloning, Growing and Induction  ==

Transform wt SM2 in <strike>pT7A3 vector (2254 pT7A3-wtSM2)</strike>

2301 pR-wtSM2-NucA 

into E coli K12 CAG597 or JM101. Grow cells in LB/amp or Kan, at 30 oC, induction at 30 oC o/n. 

=== Ecoli strains for expression  ===

'''NEB&nbsp;CAG597''' 

F- lacZ(am) pho(am) tyrT[supC(ts)] trp(am) rpsL(StrR) rpoH(am)165 zhg::Tn10 mal(am)<br>Strain Properties 

dam/dcm Methylation: dam+ , dcm+ 

Protease-deficient: Yes 

Laclq: No Drug Resistance: tet, str 

<br> 

'''Strain W3110&nbsp;'''http://cgsc2.biology.yale.edu/Strain.php?ID=5446<br>CGSC#: 4474<br>Strain Designation<br>Designation Source Preference<br>W3110 Lederberg J. 1 

Sex(Hfr,F+,F-,or F'): F-<br>3 Mutations:<br>Mutation Location Certainty Display<br>''LAM- 17.40 1 λ-<br><br>rph-1 82.20 1 rph-1'' 

<br> 

'''HB101: '''&nbsp;http://cgsc2.biology.yale.edu/Strain.php?ID=130465<br>''F- mcrB mrr hsdS20(rB- mB-) recA13 leuB6 ara-14 proA2 lacY1 galK2 xyl-5 mtl-1 rpsL20(SmR) glnV44 λ-''<br>Please note that different sources have different genotypes so treat this information with caution.<br>From a GIBCO BRL list of competent cells.<br>Hybrid of E. coli K12 and E. coli B (but 98% K strain AB266 according to Smith et al.)<br>Host for pBR322 and many plasmids<br>Sigma lists the deletion Δ(gpt,proA). Check this.<br>Promega does not list F-, mcrB, or mrr<br>Streptomycin resistant 

CGSC#: 12554<br>Strain Designation<br>Designation Source Preference<br>HB101 Boyer H. 1 

Sex(Hfr,F+,F-,or F'): F-<br>13 Mutations:<br>Mutation Location Certainty Display<br>araC14 1.52 1 araC14<br>leuB6(Am) 1.74 1 leuB6(Am)<br>DE(gpt-proA)62 5.51 1 Δ(gpt-proA)62<br>lacY1 7.79 1 lacY1<br>glnX44(AS) 14.99 1 glnX44(AS)<br>galK2(Oc) 16.99 1 galK2(Oc)<br>LAM- 17.40 1 λ-<br>recA13 60.80 1 recA13<br>rpsL20(strR) 74.84 1 rpsL20(strR)<br>xylA5 80.34 1 xylA5<br>mtl-1 81.26 1 mtl-1<br>thiE1 90.34 1 thiE1<br>hsdS20([]) 98.67 1 [hsdS20] 

<br> 

'''JM101:&nbsp;'''http://cgsc2.biology.yale.edu/Strain.php?ID=121248<br>Genotype<br>F´ traD36 proA+B+ lacIq Δ(lacZ)M15/ Δ(lac-proAB) glnV thi 

CGSC#: 12137<br>Strain Designation<br>Designation Source Preference<br>JM101 1 

Sex(Hfr,F+,F-,or F'): F'<br>3 Mutations:<br>Mutation Location Certainty Display<br>DE(gpt-lac)0 5.51 1 Δ(gpt-lac)0<br>glnX44(AS) 14.99 1 glnX44(AS)<br>thiE1 90.34 1 thiE1<br>