2. The addition of sodium pyruvate has been shown to hasten
bacterial growth on plates.
3. If gene silencing is performed for an essential gene, colonies
will not be recovered in plates. In our experience, depending
on the gene, a partial silencing is tolerated by the cells. When an
essential target gene is expected, designing sgRNA for both
strands is critical for evaluating the results. In this situation, no
colonies will be recovered when dCas9 is expressed along
sgRNA pairing with the coding strand; when sgRNA is capable
of pairing with the template strand, colonies will be recovered if
the decrease in protein levels (30–50%) is tolerated by the cells.
4. For digesting the PCR fragments referring to the sgRNA cassette with XmaI, it is recommended that at least one more
nucleotide be included at the 5
0 end of primers, right after the
XmaI site. Some restriction enzymes need additional nucleotides to recognize their sites at the end of DNA.
5. Even when ordering a synthetic gene, we recommend
re-amplifying only the sgRNA cassette. In our hands, digestion
and ligation reactions are much more efficient if the DNA is
purified directly from the reactions (PCR or digestion), with no
agarose gel steps. We use an agarose gel only to verify DNA
products, but never for product recovery. This strategy has
greatly increased our ligation efficiency.
6. For PCR with pMaOri2 primers, we have adopted a protocol in
which it is not necessary to purify genomic DNA. When
performing colony PCR in E. coli to assess sgRNA ligation
into pMaOri.dCas9, we add 1 μL of water in 0.2 mL PCR
tubes, and with the aid of a pipette, we scrape gently the surface
of the selected colony (marking below the plate the number or
letter to identify the colony) and gently mix the PCR tube.
Next, 14 μL of PCR mix are added to the samples. Positive
colonies are then again scraped off the plates and grown in
liquid medium. To evaluate L. biflexa recombinant cells, we
perform PCR of the growth cultures: we centrifuge 100 μL of
saturated culture, wash the pellet once in water, and resuspend
it in 10 μL of water, where 1 μL of the cell suspension is used as
the template for PCR. In both situations, an initial denaturation step of 5 min is established in PCR.
7. Additional washings and centrifugation steps with sterile water
in 1.5 mL microtubes ensure the elimination of salt from the
samples. Due to the small conical bottom of the tubes, leptospiral pellets, which are normally fragile, stay well sedimented,
facilitating the aspiration of residual supernatant containing
residual salt from the EMJH medium. After adopting this
protocol, we drastically reduced the loss of cuvettes.
120
Luis Guilherme Virgı ´lio Fernandes and Ana Lucia Tabet Oller Nascimento
bacterial growth on plates.
3. If gene silencing is performed for an essential gene, colonies
will not be recovered in plates. In our experience, depending
on the gene, a partial silencing is tolerated by the cells. When an
essential target gene is expected, designing sgRNA for both
strands is critical for evaluating the results. In this situation, no
colonies will be recovered when dCas9 is expressed along
sgRNA pairing with the coding strand; when sgRNA is capable
of pairing with the template strand, colonies will be recovered if
the decrease in protein levels (30–50%) is tolerated by the cells.
4. For digesting the PCR fragments referring to the sgRNA cassette with XmaI, it is recommended that at least one more
nucleotide be included at the 5
0 end of primers, right after the
XmaI site. Some restriction enzymes need additional nucleotides to recognize their sites at the end of DNA.
5. Even when ordering a synthetic gene, we recommend
re-amplifying only the sgRNA cassette. In our hands, digestion
and ligation reactions are much more efficient if the DNA is
purified directly from the reactions (PCR or digestion), with no
agarose gel steps. We use an agarose gel only to verify DNA
products, but never for product recovery. This strategy has
greatly increased our ligation efficiency.
6. For PCR with pMaOri2 primers, we have adopted a protocol in
which it is not necessary to purify genomic DNA. When
performing colony PCR in E. coli to assess sgRNA ligation
into pMaOri.dCas9, we add 1 μL of water in 0.2 mL PCR
tubes, and with the aid of a pipette, we scrape gently the surface
of the selected colony (marking below the plate the number or
letter to identify the colony) and gently mix the PCR tube.
Next, 14 μL of PCR mix are added to the samples. Positive
colonies are then again scraped off the plates and grown in
liquid medium. To evaluate L. biflexa recombinant cells, we
perform PCR of the growth cultures: we centrifuge 100 μL of
saturated culture, wash the pellet once in water, and resuspend
it in 10 μL of water, where 1 μL of the cell suspension is used as
the template for PCR. In both situations, an initial denaturation step of 5 min is established in PCR.
7. Additional washings and centrifugation steps with sterile water
in 1.5 mL microtubes ensure the elimination of salt from the
samples. Due to the small conical bottom of the tubes, leptospiral pellets, which are normally fragile, stay well sedimented,
facilitating the aspiration of residual supernatant containing
residual salt from the EMJH medium. After adopting this
protocol, we drastically reduced the loss of cuvettes.
120
Luis Guilherme Virgı ´lio Fernandes and Ana Lucia Tabet Oller Nascimento