Which Competent Cells to Use for Cloning siRNAs into pSilencer™?
Here we describe the results of transfection and sequencing using five common commercial strains of competent cells for background levels and sequence fidelity of cloned inserts. Each cell type (50 µl) was transformed with a ligation mix (3 µl) containing 100 ng pSilencer 2.0-U6 plasmid with either 8 ng of either a GAPDH siRNA hairpin insert or a 'no insert' control. The cells were transformed according to the manufacturers' protocols. All 50 µl of transformed cells were plated on LB/Ampicillin100 plates for 18 hours at 37oC. Colonies on both the sample plate and the control plate were then counted and plasmid from two colonies on each plate was isolated and sequenced.
As you can see, DH5alpha competent cells gave the lowest background of the 5 strains tested. Because there were 7.1 times as many colonies on the test plate versus the control plate, few clones should need to be sequenced to identify a clone with the correct insert.
The sequencing results confirmed this assessment - a correct GAPDH siRNA insert was detected in all 10 clones sequenced. Both strands of the plasmid in the region of the insert were sequenced and 10% DMSO was included in all sequencing reactions to alleviate any problems sequencing through secondary structure.
At Ambion, we routinely use DH5alpha cells (Invitrogen) for cloning small inserts into our pSilencer vectors, although all of the bacterial strains tested here also worked well in the experiment. There are many other competent cells commercially available that are likely to work as well.