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| ='''Making Cells Competent'''=
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| ''Time: O’N then 3 hr next day''
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| ''Reference: Curr. Prot. in Molec. Bio, Vol. 1, 1.8.1 <nowiki>"Trans. Using CaCl2"</nowiki>''
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| Makes 64x50μL aliquots
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| ===<font color="blue">Media/Reagents</font>===
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| *LB
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| *Cells (from plate)
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| *CaCl2 Solution
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| *Dry Ice (2nd floor Althouse or S. Frear)
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| ===<font color="red">Pre</font>===
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| *Prepare CaCl2 solution<sup>1</sup>
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| *Autoclave centrifuge bottles/tubes and chill on ice
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| *Before centrifuge steps, make sure centrifuge is on and at 0°C
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| ===<font color="green">Competent Cells: Day 0</font>===
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| #Streak cells on LB plate
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| #Grow overnight at 37°C
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| ===<font color="green">Competent Cells: Day 1 (optional)</font>===
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| #Inoculate overnight starter culture (2 mL) w/ colony from plate
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| ===<font color="green">Competent Cells: Day 2</font>===
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| #Inoculate 80 mL<sup>2</sup> with appropriate amount of O’N to obtain OD6000~0.1. Place cells on ice at OD600=0.375 (do not allow growth past an OD of 0.4, as this decreases subsequent transformation efficiency). Typically this will take 40-60 min (check every 5 min when OD is getting near 0.375).
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| #Ice for 10 min.
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| #Transfer to a pre-chilled centrifuge tube (40mL) and spin at 1600g/7 min/0°C/no brake in small, cold rotor.
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| #Decant supernatant & gently resuspend pellet in 8 mL of ice-cold CaCl2 buffer.
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| #Spin 1100g/5 min/0°C.
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| #Gently resuspend pellet in 8 mL ice-cold CaCl2. Let stand on ice for 30 min.
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| #Spin 1100g/5 min/0°C. Decant supernatant & resuspend <u>''well''</u> in 1.6 mL ice-cold CaCl2. Cells may stand on ice for 12-24 hrs to increase competency.
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| #Swirl tube(s) to mix, & aliquot 50 μL into cold eppendorfs. Immerse immediately in crushed dry ice.
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| #Transfer to box(es) and store in -80°C freezer (downstairs on 2nd floor).
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| <sup>1</sup>CaCl2(per L)
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| *20 g bactotriptone
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| *5 g bacto-yeast extract
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| *0.5 g NaCl
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| *0.19 g KCl
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| *Adjust to pH 7.0 w/ NaOH
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| *Autoclave
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| *Add filter-sterilized MgCl2, MgSO4 solution to give final Mg2+ conc. of 20 mM (i.e. 10 mM MgCl2, 10 mM MgSO4)
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| <sup>2</sup>Procedure can be scaled up or down as necessary
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