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Team:UCL/protocols
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<h1>Protocols</h1> | <h1>Protocols</h1> | ||
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| + | <h4>Creating Competent Cells</h4> | ||
| + | |||
| + | <p><b>Materials</b><br/> | ||
| + | LB Media, 50ml Falcon Tubes, Ice, Chilled centrifuge, Calcium Chloride (CaCl2), Eppendorf tubes (300ul/tube)<br/><br/> | ||
| + | |||
| + | <b>Procedure</b><br/> | ||
| + | 1. Inoculate a single colony into 5ml Lb in 50ml falcon tube. Grown O/N @ 37oC<br/> | ||
| + | 2. Use 1ml to inoculate 100ml of LB in 250ml bottle the next morning.<br/> | ||
| + | Shake @ 37oC for 1.5-3 hours.<br/><br/> | ||
| + | |||
| + | Or<br/><br/> | ||
| + | |||
| + | 1. Inoculate a single colony into 25ml LB in a 250ml bottle in the morning<br/> | ||
| + | 2. Shake @ 37oC for 4-6 hours.<br/><br/> | ||
| + | |||
| + | Then…<br/><br/> | ||
| + | |||
| + | 3. Put the cells on ice for 10mins (keep cold from now on).<br/> | ||
| + | 4. Collect the cells by centrifugation in the big centrifuge for 3 minutes @ 6Krpm.<br/> | ||
| + | 5. Decant supernatant and gently resuspend on 10ml cold 0.1M CaCl (cells sensitive to mechanical disruption).<br/> | ||
| + | 6. Incubate on ice x 20 minutes<br/> | ||
| + | 7. Centrifuge as in 2.<br/> | ||
| + | 8. Discard supernatant and gently resuspend on 5ml cold 0.1M CaCl/15%Glycerol.<br/> | ||
| + | 9. Dispense in microtubes (300ųl/tube). Freeze at -80oC.<br/> | ||
| + | |||
| + | </p> | ||
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| + | <br/> | ||
| + | <h4>Transformation of competent cells</h4> | ||
| + | |||
| + | <p> | ||
| + | <b>Materials</b><br/> | ||
| + | Competent Cells, Plasmid DNA, Antibiotic Plates<br/><br/> | ||
| + | |||
| + | <b>Procedure</b><br/> | ||
| + | |||
| + | 1. T haw competent cells on ice<br/> | ||
| + | 2. 50uL cells enough for 1 transformation<br/> | ||
| + | 3. Add 1ug of DNA to 50uL competent cells<br/><br/> | ||
| + | |||
| + | If biobrick from distribution, resuspend DNA well in 10uL ddH20<br/><br/> | ||
| + | |||
| + | 4. Add 1uL biobrick DNA to 50uL competent cells<br/> | ||
| + | 5. Add 1uL RFP control to 50uL competent cells for your control transformation<br/> | ||
| + | 6. Flick by hand or pipette up and down gently<br/> | ||
| + | 7. Place cells on ice for 30 minutes<br/> | ||
| + | 8. Place cells in water bath at 42oC for 40 seconds<br/> | ||
| + | 9. Place cells on ice for 2 minutes<br/> | ||
| + | 10. Add 0.5mL of LB media and place in incubator for a maximum of 2 hours (37oC/250rpm)42oC | ||
| + | (200 µl SOC media can be used to improve transformation efficiency)42oC | ||
| + | 11. Label two petri dishes with LB agar and the appropriate antibiotics(s) with the part number, plasmid backbone and antibiotic resistance<br/> | ||
| + | 12. Plate 50 µl and 500 µl of the transformation onto the dishes, and spread.<br/> | ||
| + | 13. Incubate the plates at 37ºC for 12-14 hours, making sure the agar side of the plate is up.<br/><br/> | ||
| + | |||
| + | If incubated for too long the antibiotics start to break down and un-transformed cells will begin to grow. This is especially true for ampicillin - because the resistance enzyme is excreted by the bacteria, and inactivates the antibiotic outside of the bacteria<br/><br/> | ||
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| + | You can pick a single colony, make a glycerol stock, grow up a cell culture and miniprep.<br/><br/> | ||
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| + | Count the colonies on the 20 μl control plate and calculate your competent cell efficiency.<br/> | ||
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| + | <p> | ||
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</div> | </div> | ||
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Latest revision as of 14:54, 5 September 2014
