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| ==RbCl Competent Cell Prep==
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| ====Solutions and Supplies====
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| *sterilized 250 ml centrifuge bottles <br>
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| *sterilized 1.5 ml microfuge tubes (at least 50)
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| *sterilized 100 ml LB in 250ml flask
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| *filter sterilized 100 ml chilled RF1 per 300 ml ''E. coli'' culture (or 33 ml per 100 ml culture)
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| *filter sterilized 12 ml chilled RF2 per 300 ml ''E. coli'' culture (or 4 ml per 100 ml culture)
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| =====RF1=====
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| {| {{table}}
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| | align="center" style="background:#f0f0f0;"|'''Combine for 1 L:'''
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| | align="center" style="background:#f0f0f0;"|
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| | align="center" style="background:#f0f0f0;"|'''[final]'''
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| |-
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| | Rubidium Chloride (RbCl, sigma R2252)||12.1 g||100 mM
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| |-
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| | Manganese(II) chloride tetrahydrate (MnCl<sub>2</sub> 4H<sub>2</sub>O, sigma M3634)||9.9 g||50 mM
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| |-
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| | Potassium acetate||2.94g or 30 ml of 1M stock, pH 7.5||30 mM
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| |-
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| | Calcium chloride dihydrate (CaCl<sub>2</sub> 2H<sub>2</sub>O)||1.48 g||10 mM
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| |-
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| | Glycerol||150 g or 120ml||15% wt/vol
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| |}
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| *Adjust final pH to 5.8 using 0.2M acetic acid (maybe 400ul for 33ml). Filter-sterilize.
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| *Glacial acetic acid: 1.049g cm-3 / 60.05g mol-1 = 17.47 M
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| =====RF2=====
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| {| {{table}}
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| | align="center" style="background:#f0f0f0;"|'''Combine for 1 L:'''
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| | align="center" style="background:#f0f0f0;"|
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| | align="center" style="background:#f0f0f0;"|'''[final]'''
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| |-
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| | MOPS (sigma M1254)||2.1g||10 mM
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| |-
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| | Rubidium Chloride (RbCl, sigma R2252)||1.2 g||10 mM
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| |-
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| | Calcium chloride dihydrate (CaCl<sub>2</sub> 2H<sub>2</sub>O)||11 g||75 mM
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| |-
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| | Glycerol||150 g or 120ml||15% wt/vol
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| |}
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| *Adjust final pH to 6.8 using 1M NaOH (maybe 200ul for 30ml). Filter-sterilize.
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| ====Cell Preparation Procedure====
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| =====Day 1=====
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| *Streak DH5alpha from frozen glycerol stock on the LB plate.
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| *Incubate at 37oC, over night.
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| *Prepare sterilized LB.
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| =====Day 2=====
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| *Pick up a single colony from the LB plate.
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| *Inoculate to 3ml sterilized LB.
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| *Incubate at 37oC, over night.
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| *Put RF1, RF2, centrifuge tube and eppendorf tubes into the 4oC refrigerator.
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| =====Day 3=====
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| *Put RF1, RF2, centrifuge tube and eppendorf tubes on ice.
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| *Inoculate 1ml of over night culture to 100ml of LB in flask.
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| *Monitor OD600 from initial until 0.2 to 0.6. [0.4 to 0.55 optimum]
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| *Transfer culture to centrifuge bottle and chill on ice 10 -15 min.
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| *Pellet cells by centrifugation at 2700g (4200rpm in an F14 6x250y rotor) for 10 min at 4oC.
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| *Decant liquid and stand the bottle in an inverted position for < 1 min.
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| *resuspend in 1/3 original volume (33 ml) chilled RF1 buffer gently.
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| *Optimally, resuspend using a 25-ml disposable pipet.
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| **RbCl will permanently stain glass pipets.
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| **Note: resuspend gently, DO NOT VORTEX.
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| ***Want to maintain pili structures on surface of E coli cell.
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| *Continue mixing until cells are evenly resuspended and no clumps are visible.
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| *Incubate cells/RF1 on ice for 15 min.
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| *Pellet cells by centrifugation at 580g (1950rpm in an F14 6x250y rotor) for 15 min at 4oC.
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| *Decant liquid and gently resuspend in 1/25 original volume (4 ml) chilled RF2 buffer.
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| *Incubate cells/RbCl buffer 2 on ice for 15min.
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| *Get eppendor tubes and box ready.
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| *Aliquot 100 ul each into chilled 1.5 ml eppendorf tubes and freeze on dry ice (or ice).
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| *Store at -80oC.
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| ====Determine transformation efficiency====
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| *Dilute control plasmid DNA (known DNA conc) to 1 ng/ul and transform using 1 ul.
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| *Thaw competent cells on ice.
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| *Compare previous lot to current lot
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| *Combine 1ul of diluted pDNA and 100 ul competent cells.
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| *Incubate on ice 30-60 min (40 min optimum).
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| *Heat shock at 42oC for 90 sec, place on ice 5 - 15 min.
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| *Add 900ul LB and incubate at 37oC for 30-60 min (45 min optimum).
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| *Plate 100 ul onto antibiotic plate.
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| *Dilute the culture 10 times and plate 100 ul onto antibiotic plate.
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| *Dilute the culture 100 times and plate 100 ul onto antibiotic plate.
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| *Count the numbers of colonies in the next morning.
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