recovery is best if Leptospira concentration was at least
10
7 cells/mL prior to filtration.
37. Once transposon is cis-inserted into genome, it is not spontaneously lost. Therefore, kanamycin selection is not necessary
after initial growth in EMJH liquid media with 40 μg/mL of
kanamycin.
38. It is typically considered that the concentration of Tween-80
and glycerol present in EMJH liquid media is sufficient for
long-term storage in cryogenic vials without adding additional
cryoprotectant. Some laboratories use 5% DMSO as a cryoprotectant [47]. Our laboratory typically makes up two freezer
tubes for each Tn mutant for long-term storage at À80
C: one
with 30% glycerol added and one without. We have not noticed
any difference in growth between those stored with and without glycerol during resurrection.
39. It is highly recommended to share the Tn library with a collaborator. This will provide an off-site backup of your Tn library in
case of catastrophic equipment failure.
40. Spot PCR is less reliable for this procedure; we recommend
using a genomic DNA purification kit or an automated genomic DNA purification system, such as PureLink or Maxwell
16, respectively.
41. We typically run at least two different reactions at once to save
on time (e.g., TnK1 with Deg1, TnK2, and Deg1). One will
typically amplify. If nested PCR does not yield multiple bands
after round 2, we will try the other combinations.
42. Round 1 does not typically generate visible bands by gel electrophoresis. Therefore, it is not recommended to attempt visualization by electrophoresis from round 1 product.
43. Match TnK1 to TnKN1 and TnK2 to TnKN2.
44. We use RedSafe in lieu of ethidium bromide as an intercalating
fluorescent dye and get excellent and well-resolved visualization of bands.
45. The multiple bands visualized by electrophoresis do not infer
multiple insertion sites. Instead, since oligo 2 is a semi-random/degenerate primer [41], it is annealing to several locations upstream of the transposon insertion site, resulting in the
appearance of multiple bands (Fig. 7). Previous research confirmed multiple banding is due to single-site transposon insertion [16]. Therefore, the PCR product can be sent for
sequencing without single-band gel extraction.
46. Since some companies use enzymatic purification of PCR products which may impact multiband PCR products, we occasionally complete PCR cleanup by column purification before
sending products for sequencing. With Genewiz, we have
Random Transposon Mutant Library
93
10
7 cells/mL prior to filtration.
37. Once transposon is cis-inserted into genome, it is not spontaneously lost. Therefore, kanamycin selection is not necessary
after initial growth in EMJH liquid media with 40 μg/mL of
kanamycin.
38. It is typically considered that the concentration of Tween-80
and glycerol present in EMJH liquid media is sufficient for
long-term storage in cryogenic vials without adding additional
cryoprotectant. Some laboratories use 5% DMSO as a cryoprotectant [47]. Our laboratory typically makes up two freezer
tubes for each Tn mutant for long-term storage at À80
C: one
with 30% glycerol added and one without. We have not noticed
any difference in growth between those stored with and without glycerol during resurrection.
39. It is highly recommended to share the Tn library with a collaborator. This will provide an off-site backup of your Tn library in
case of catastrophic equipment failure.
40. Spot PCR is less reliable for this procedure; we recommend
using a genomic DNA purification kit or an automated genomic DNA purification system, such as PureLink or Maxwell
16, respectively.
41. We typically run at least two different reactions at once to save
on time (e.g., TnK1 with Deg1, TnK2, and Deg1). One will
typically amplify. If nested PCR does not yield multiple bands
after round 2, we will try the other combinations.
42. Round 1 does not typically generate visible bands by gel electrophoresis. Therefore, it is not recommended to attempt visualization by electrophoresis from round 1 product.
43. Match TnK1 to TnKN1 and TnK2 to TnKN2.
44. We use RedSafe in lieu of ethidium bromide as an intercalating
fluorescent dye and get excellent and well-resolved visualization of bands.
45. The multiple bands visualized by electrophoresis do not infer
multiple insertion sites. Instead, since oligo 2 is a semi-random/degenerate primer [41], it is annealing to several locations upstream of the transposon insertion site, resulting in the
appearance of multiple bands (Fig. 7). Previous research confirmed multiple banding is due to single-site transposon insertion [16]. Therefore, the PCR product can be sent for
sequencing without single-band gel extraction.
46. Since some companies use enzymatic purification of PCR products which may impact multiband PCR products, we occasionally complete PCR cleanup by column purification before
sending products for sequencing. With Genewiz, we have
Random Transposon Mutant Library
93