Protocol 11 - A. Transformation of Max Efficiency DH5 Competent Bacteria with
Ligated Bluescript© Phagemids; B. Plating of Transforms on LB/AMP/X-gal/IPTG
Plates
Supplies needed:
- Max Efficiency DH5 Competent Bacteria
- 17 X 100 mm polypropylene tubes
- pUC19 control plasmid
- TE solution in 1.5 ml microfuge tubes
- Ligation reactions with Bluescript© Phagemid
- SOC medium
- LB/AMP/X-gal plates
- 40 mM IPTG
Equipment needed:
- 42°C water bath
- wet ice
- P-10, 100, 1000 micropipettors
- Incubator shaker at 37°C
Important Notes to Remember:
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Calculate the volume of cells you will need and use/thaw only that amount that
you require to avoid thawing/re-thawing of cells which lowers transformation
efficiency.
Use a different pipette tip for each component of the reaction so as to NOT
contaminate the stock solutions.
As usual, it is very important that you employ sterile technique to avoid
contamination.
Only remove the enzyme from the freezer or from the ice when you are prepared
to add it. When any enzyme is off the ice for a prolonged period, it can lose
activity over time if it is not on ice or in the freezer. Minimize the time it is
unfrozen.
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1. Be sure that water bath is set to 42°C for heat-shock of cells at later step.
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2. Thaw the competent Max Efficiency DH5 Competent Bacteria on wet ice.
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3. Place the appropriate number (depending on number of different ligations to be
performed plus one control) of 17 X 100 mm polypropylene tubes on ice.
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4. Once the cells have thawed on wet ice, gently mix the cells to resuspend.
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5. Add 100 μl of the competent cells into the chilled polypropylene tubes. (For a
control, add 5 μl of pUC19 to one of the tubes with competent cells.)
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6. Gently pipette the cells up and down and tap the tube gently to mix.
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7. Take the ligation reaction and dilute 5X in TE (e.g. take 10 μl of ligation
reaction and dilute to final volume of 50 μl in TE)
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8. Add 1 μl (approximately 1-10 ng DNA fragments) to the cells.
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9. Gently pipette the cells up and down and tap the tube gently to mix.
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10. Incubate on ice for exactly 30 minutes.
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11. Heat shock the cells for exactly 45 seconds in the 42°C water bath.
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12. Place the cells on ice for exactly 2 minutes.
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13. Add 0.9 ml (900 μl) of SOC medium to the cells.
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14. Place the cells on a shaker and shake at 37°C for one hour or overnight.
Plating the Transforms on LB/AMP/X-Gal/IPTG plates:
To prepare the LB/AMP/X-gal plates for plating, you will need to spread, using sterile
technique, 100 μl of 40 mM IPTG solution onto the plates.
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15. Control plate - Dilute the "control" (pUC19) transformed cells 1:100 in SOC
medium (e.g. 1 μl into 99 μl of SOC medium) and then spread 100 μl onto
LB/AMP/X-gal/IPTG plates using sterile technique.
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16. Experimental Plates - Dilute the fragment-inserted transformed cells 1:100 in
SOC medium (e.g. 1 μl into 99 μl of SOC medium) and then spread 100 μl onto
LB/AMP/X-gal/IPTG plates using sterile technique.