TABLE 1

The effect of changing different reaction parameters on the in vitro transposition frequency of Himar1

Reaction parameteraAbsolute transposition frequencybRelative transposition frequencyc
Time (min)
30 (5)0.35 (±0.07)0.02 (±0.004)
60 (4)2.02 (±0.76)0.12 (±0.05)
180 (3)5.61 (±0.35)0.34 (±0.02)
360 (5)16.69 (±3.40)1.0 (±0.20)
Temperature
15.5° (4)0.94 (±0.13)0.11 (±0.02)
23° (4)3.83 (±0.48)0.45 (±0.06)
28° (4)8.52 (±0.49)1.0 (±0.06)
35° (3)6.02 (±0.42)0.71 (±0.05)
40° (4)3.63 (±0.21)0.42 (±0.02)
MgCl2 (mM)
0 (4)0.000.00
5 (3)5.45 (±0.90)0.82 (±0.14)
10 (4)5.64 (±0.89)1.00 (±0.13)
15 (4)5.68 (±0.96)0.86 (±0.14)
Transposase (nM)
0.6 (3)0.19 (±0.06)0.03 (±0.01)
1.2 (3)0.87 (±0.05)0.14 (±0.01)
6.0 (3)6.39 (±0.57)1.0 (±0.09)
15 (2)5.85 (±0.67)0.92 (±0.12)
60 (3)1.0 (±0.06)0.16 (±0.01)
150 (3)0.64 (±0.09)0.10 (±0.01)
300 (3)0.20 (±0.06)0.03 (±0.01)
600 (3)0.05 (±0.01)0.01 (±0.001)
Transposon size (kb)
1.3 (4)17.97 (±4.27)1.0 (±0.10)
2.3 (12)14.19 (±2.40)0.82 (±0.14)
3.8 (5)3.10 (±0.42)0.18 (±0.02)
5.7 (3)1.53 (±0.14)0.09 (±0.01)
7.3 (3)1.17 (±0.50)0.07 (±0.03)
Donor flanking DNA
pMMKan (4)4.34 (±0.58)1.0 (±0.13)
pMM26 (4)10.47 (±0.60)2.40 (±0.14)
pMM32 (4)5.93 (±0.26)1.36 (±0.06)
  • a Numbers in parentheses indicate the number of replicates for that treatment.

  • b Average absolute transposition frequency. Absolute frequencies for each parameter are calculated by dividing the number of Kanr-Ampr colonies produced in the in vitro transposition reaction by the number of Ampr colonies and multiplying by 1000. The average is the sum of the individual frequencies for each parameter divided by the number of replicates. Each error is the standard error of the mean, which is calculated as the parameter standard deviation divided by the square root of the number of replicates.

  • c Relative frequencies are calculated by dividing each of the absolute frequencies in a given parameter group by the average absolute maximum transposition frequency within that group. In the case of the donor flanking DNA data set, the absolute transposition frequency for the donor containing the flanking DNA used for all other reactions, pMMKan, was used for normalization. Relative errors are computed by dividing the absolute errors within a group by the maximum average absolute frequency within that group.