Editing Promega Protein Expression (section)

== S30 T7 High-Yield Protein Expression System Protocol ==

<span style="font-size:larger;">1, Remove reagents from freezer and thaw on ice (DNA tempplate, S30 Premix Plus, S30 Extract Circular, Nuclease-Free water)</span>

<span style="font-size:larger;">aliquot 18</span><span style="color: rgb(34, 34, 34); font-family: Roboto, arial, sans-serif; font-size: 15.6px;">μl</span><span style="font-size:larger;">&nbsp;<span style="color: rgb(34, 34, 34); font-family: Roboto, arial, sans-serif;">T7 S30 Extract, to 500</span></span><span style="color: rgb(34, 34, 34); font-family: Roboto, arial, sans-serif; font-size: 15.6px;">μl tube, store at - 80 oC freezer.</span><span style="color: rgb(34, 34, 34); font-family: Roboto, arial, sans-serif; font-size: 15.6px;">&nbsp;</span><span style="font-size:larger;"></span>

<span style="font-size:larger;">2, In a 1.5 mL centrifuge tube, standard reaction:</span>

<span style="font-size:larger;">DNA template </span><span style="color: rgb(34, 34, 34); font-family: Roboto, arial, sans-serif; font-size: 15.6px;">Circular,&nbsp;</span><span style="font-size:larger;">1<span style="color: rgb(34, 34, 34); font-family: Roboto, arial, sans-serif;">μg</span></span>

<span style="font-size:larger;"><span style="color: rgb(34, 34, 34); font-family: Roboto, arial, sans-serif;">S30 Premix Plus,&nbsp; 20</span></span><span style="color: rgb(34, 34, 34); font-family: Roboto, arial, sans-serif; font-size: 15.6px;">μl</span>

<span style="font-size:larger;"><span style="color: rgb(34, 34, 34); font-family: Roboto, arial, sans-serif;">T7 S30 Extract, 18&nbsp;</span></span><span style="color: rgb(34, 34, 34); font-family: Roboto, arial, sans-serif; font-size: 15.6px;">μl&nbsp;</span>

<span style="font-size:larger;"><span style="color: rgb(34, 34, 34); font-family: Roboto, arial, sans-serif;"></span></span><span style="font-size:larger;"><span style="color: rgb(34, 34, 34); font-family: Roboto, arial, sans-serif;">Add Nuclease-Free water to&nbsp;final volume of 50&nbsp;</span></span><span style="color: rgb(34, 34, 34); font-family: Roboto, arial, sans-serif; font-size: 15.6px;">μl in total</span>

<span style="color: rgb(34, 34, 34); font-family: Roboto, arial, sans-serif; font-size: 15.6px;">3, Pipette several times to mix</span>

<span style="color: rgb(34, 34, 34); font-family: Roboto, arial, sans-serif; font-size: 15.6px;">4, Centrifuge for 5 seconds</span>

<span style="color: rgb(34, 34, 34); font-family: Roboto, arial, sans-serif; font-size: 15.6px;">5, Put into 37 degree shaker overnight</span>

<span style="color: rgb(34, 34, 34); font-family: Roboto, arial, sans-serif; font-size: 15.6px;">6, Place in ice for 2&nbsp;min. Add 50 ul cold L</span><span style="color: rgb(34, 34, 34); font-family: Roboto, arial, sans-serif; font-size: 15.6px;">ysis buffer.</span>

<span style="color: rgb(34, 34, 34); font-family: Roboto, arial, sans-serif; font-size: 15.6px;">7, Transfer 50 ul of 10% Nickel magnetic beads to a tube,&nbsp; put on a magnetic rack,&nbsp; remove suspension buffer&nbsp;&nbsp;using 200 ul pipet tip.</span>

<span style="color: rgb(34, 34, 34); font-family: Roboto, arial, sans-serif; font-size: 15.6px;">Add in vitro translation reaction solution, mix, incubate on ice, mix gently by swireling, mix gentely every 30 seconds, incubate for 5 min for efficient binding<font color="#222222" face="Roboto, arial, sans-serif"><span style="font-size: 15.6px;"></span></font>, put on a magnetic rack, transfer&nbsp;liquid to another tube using 200 ul pipet tip</span><font color="#222222" face="Roboto, arial, sans-serif"><span style="font-size: 15.6px;"></span></font>

<span style="color: rgb(34, 34, 34); font-family: Roboto, arial, sans-serif; font-size: 15.6px;">8, Wash 3 X 200 ul&nbsp; of&nbsp;Lysis Buffer, put on a magnetic rack, remove liquid using 200 ul pipet tip</span>

<span style="color: rgb(34, 34, 34); font-family: Roboto, arial, sans-serif; font-size: 15.6px;">9, Wash 1 X Washing Buffer, put on a magnetic rack, remove liquid using 200 ul pipet tip</span>

<span style="color: rgb(34, 34, 34); font-family: Roboto, arial, sans-serif; font-size: 15.6px;">10, Add 25 ul Buffer B, mix,&nbsp;&nbsp;incubate on ice for 1 min</span>

<span style="color: rgb(34, 34, 34); font-family: Roboto, arial, sans-serif; font-size: 15.6px;">11, Put onto magnetic rack, transfer liquid to new tube.</span>

<span style="color: rgb(34, 34, 34); font-family: Roboto, arial, sans-serif; font-size: 15.6px;">12, repeat step 10, 11, combine liquid</span>

<span style="color: rgb(34, 34, 34); font-family: Roboto, arial, sans-serif; font-size: 15.6px;">13. Measure protein concentration with nanodrop, using Buffer B as blank ,&nbsp;usually 2 mg/ml</span>

<span style="color: rgb(34, 34, 34); font-family: Roboto, arial, sans-serif; font-size: 15.6px;">14, Add equal volume of cold 80% glycerol, mix gentely,&nbsp;</span>

<span style="color: rgb(34, 34, 34); font-family: Roboto, arial, sans-serif; font-size: 15.6px;">&nbsp; optionally, aliquot 10 ug to clean tubes.</span>

<span style="color: rgb(34, 34, 34); font-family: Roboto, arial, sans-serif; font-size: 15.6px;">15, Print label and store protein in -80 oC freezer</span>





'''<span style="color: rgb(34, 34, 34); font-family: Roboto, arial, sans-serif; font-size: 15.6px;">L</span><span style="color: rgb(34, 34, 34); font-family: Roboto, arial, sans-serif; font-size: 15.6px;">ysis Buffer:</span>'''

<span style="color: rgb(34, 34, 34); font-family: Roboto, arial, sans-serif; font-size: 15.6px;">0.5% NP-40, 10 mM Imidazole, 0.5 M NaCl, 20 mM Tris-HCl, pH 8.0</span>

'''<span style="color: rgb(34, 34, 34); font-family: Roboto, arial, sans-serif; font-size: 15.6px;">Washing Buffer:&nbsp;&nbsp;</span>'''

<span style="color: rgb(34, 34, 34); font-family: Roboto, arial, sans-serif; font-size: 15.6px;">1.5 M</span><span style="color: rgb(34, 34, 34); font-family: Roboto, arial, sans-serif; font-size: 15.6px;">&nbsp;</span><span style="color: rgb(34, 34, 34); font-family: Roboto, arial, sans-serif; font-size: 15.6px;">NaCl, 20 mM T</span><span style="color: rgb(34, 34, 34); font-family: Roboto, arial, sans-serif; font-size: 15.6px;">ris-HCl, pH8.0</span>

'''<span style="color: rgb(34, 34, 34); font-family: Roboto, arial, sans-serif; font-size: 15.6px;">Buffer B:</span>'''

<span style="color: rgb(34, 34, 34); font-family: Roboto, arial, sans-serif; font-size: 15.6px;">10% glycerol, 500 mM Imida</span><span style="color: rgb(34, 34, 34); font-family: Roboto, arial, sans-serif; font-size: 15.6px;">zole, 0.5 M NaCl, 1 mM DTT, 20 mM Tris-HCl, pH8.0</span>