4/1/2015
ClpX,ClpB and ChiT + pJH1
One Shot® Mach1™-T1R Chemically Competent E. coli
Transforming Competent
Cells
Perform the following before starting the transformation procedure:
• Equilibrate a water bath to 42°C.
• Warm the vial of S.O.C. Medium (supplied with the kit) to room temperature.
• Spread X-Gal onto LB agar plates containing antibiotic, if desired.
• Warm the selective plates in a 37°C incubator for 30 minutes (use one plate for each transformation).
If you are including
the pUC19 control, make sure that you have one LB agar plate containing 100 µg/ml ampicillin.
Note: For optimal growth
of Mach1™-T1R E. coli cells, it is essential that selective plates are prewarmed to 37°C prior to spreading.
Transformation Procedure
We recommend including the pUC19 control plasmid DNA supplied with the kit in your transformation experiment to verify
the efficiency of the competent cells.
1. Thaw, on ice, one vial of One Shot® Mach1™-T1R Chemically Competent E. coli for each transformation.
2. Add 1 to 5 µl of the DNA (10 pg to 100 ng) into a vial of One Shot® cells and mix gently. Do not mix by pipetting up and
down. If you are transforming the pUC19 control, add 1 µl (10 pg) into a separate vial of One Shot® cells and mix gently.
3. Incubate the vial(s) on ice for 30 minutes.
4. Heat-shock the cells for 30 seconds at 42°C without shaking.
5. Remove the vial(s) from the 42°C bath and place them on ice for 2 minutes.
6. Add 250 µl of room temperature S.O.C. Medium to each vial.
7. Cap the vial(s) tightly and shake horizontally at 37°C for 1 hour at 225 rpm in a shaking incubator.
8. Spread 25-100 µl of the transformation mix on a prewarmed selective plate. Store the remaining transformation mix at
+4°C. Additional cells may be plated out the next day, if desired.
9. Invert the plate(s) and incubate at 37°C. If you are using ampicillin selection, visible colonies should appear within 8 hours,
and blue/white screening can be performed after 12 hours. If you are selecting transformants with an antibiotic other than
ampicillin, incubate plates overnight.
10. Select overnight-grown colonies and analyze by plasmid isolation, PCR, or sequencing.
For plasmid isolation, inoculate a
single, overnight-grown colony in 2 ml of prewarmed selective media .
For optimal results, we recommend inoculating as much of the single colony as possible. Shake at 37°C for 4 hours
before isolating the plasmid.
Result
1-ClpX + pJH1 ,on LB+ Hygromycine (+)
2-ClpB + pJH1 , on LB+ Hygromycine(+)
3- ChiT + pJH1, on LB+ Hygromycine (+)
4- PUC 19 on LB+ Ampenicillin, (+) positive control.
5- DH5 alfa on LB+ Ampenicillin (-) negative control.
6-PUC 19 and DH5 alfa on LB+ Hygromycine (-)
