Bio-Rad Ligation and Transformation Module - Manual (Page 48)

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Note: If you are performing the ligation and transformation steps on the
same day, use the microcentrifuge containing 5 µl of the ligation mixture
that was prepared and labeled at the end of the ligation step.
16. If not already done, pipet 5 µl of the ligation reaction from the previous
stage into the "gene TF" microcentrifuge tube. Store any remaining lig-
ation reaction at 4°C or –20°C.
17. (Optional) If performing a positive control transformation, pipet 1 µl of
the positive control plasmid into an appropriately labeled microcentrifuge
tube and keep on ice.
18. Using a fresh tip, pipet 50 µl of competent bacteria directly into the
ice-cold "gene TF" tube containing 5 µl of your ligation and gently pipet
up and down two times to mix.
19. (Optional) If performing a positive control transformation, pipet 50 µl of
competent bacteria directly into the ice-cold positive control plasmid
tube and gently pipet up and down two times to mix.
20. Incubate the transformations for 10 min on ice.
21. Retrieve LB Amp IPTG agar plates from the 37°C incubator.
22. Pipet the entire volume of each transformation onto the corresponding
labeled LB Amp IPTG agar plate, and using an inoculation loop or a
sterile spreader, very gently spread the bacteria around the plate —
remember that the bacteria are still very fragile! Once the plate is
covered, stop spreading. Do not spread for more than 10 sec.
It is vital that the LB Amp IPTG agar plate be warm at this step to ensure
sufficiently high transformation efficiency. Spreading the plate until it is dry
will also reduce transformation efficiency.
Ice