51
9. Incubate the sample at 37 °C for 1 h.
10. Plate dilutions of the cells on 10 cm dishes and plate the
remainder on 245 × 245 mm plate bioassay dishes with LB
agar (see Note 26).
11. Incubate the plates at 37 °C for 12–18 h.
12. The next day, count the number of colonies on the 10 cm dishes
to determine the number of transformants (see Note 27).
13. You can further confirm the diversity of this “naive” domain
insertion library by performing colony PCR on several transformants and/or growing 10 mL LB cultures of samples, purifying
their DNA and analyzing the DNA sequence (see Note 28).
4 Notes
1. We have found that the Qiagen Miniprep kits have better yield
and less genomic DNA contamination compared to the
Midiprep or Maxiprep kits.
2. This working solution is good for at least 3 months.
3. If chilled on ice, solids may fall out of solution. If this happens,
a fresh diluent must be prepared.
4. The slowest migrating band is nicked open plasmid. The fastest migrating band is supercoiled DNA and the band in the
middle is linear DNA.
5. The optimal DNase I concentration is the one that produces a
sharp band of linearized plasmid. To achieve this, 30–50% of
the plasmid usually remains supercoiled. Overdigestion of the
plasmid will result in smearing indicating deletions at the ends
of the digestion products.
6. The 30 s staggered intervals are to insure that each tube will be
stopped at exactly 8 min. We recommend that experiments
be performed on the same day as the optimization. If the
reactions will be completed on a different day, prepare fresh
DNase dilutions.
7. The prepared DNA can be stored at −20 °C.
8. This step creates blunt ends by filling in the end or by chewing
away any overhanging DNA. Note the amount of T4 DNA
Ligase and T4 DNA Polymerase should be calculated based on
the amount of linear DNA.
9. Do not load more than 2.5 μg per lane (of the medium-sized
lanes). If possible, do not use UV light or ethidium bromide as
these can cause DNA damage.
10. Your primers should adhere to the following guidelines as
much as possible: (1) 40–60% GC, (2) at least 18 nucleotides
Engineering Protein Switches by Domain Insertion
9. Incubate the sample at 37 °C for 1 h.
10. Plate dilutions of the cells on 10 cm dishes and plate the
remainder on 245 × 245 mm plate bioassay dishes with LB
agar (see Note 26).
11. Incubate the plates at 37 °C for 12–18 h.
12. The next day, count the number of colonies on the 10 cm dishes
to determine the number of transformants (see Note 27).
13. You can further confirm the diversity of this “naive” domain
insertion library by performing colony PCR on several transformants and/or growing 10 mL LB cultures of samples, purifying
their DNA and analyzing the DNA sequence (see Note 28).
4 Notes
1. We have found that the Qiagen Miniprep kits have better yield
and less genomic DNA contamination compared to the
Midiprep or Maxiprep kits.
2. This working solution is good for at least 3 months.
3. If chilled on ice, solids may fall out of solution. If this happens,
a fresh diluent must be prepared.
4. The slowest migrating band is nicked open plasmid. The fastest migrating band is supercoiled DNA and the band in the
middle is linear DNA.
5. The optimal DNase I concentration is the one that produces a
sharp band of linearized plasmid. To achieve this, 30–50% of
the plasmid usually remains supercoiled. Overdigestion of the
plasmid will result in smearing indicating deletions at the ends
of the digestion products.
6. The 30 s staggered intervals are to insure that each tube will be
stopped at exactly 8 min. We recommend that experiments
be performed on the same day as the optimization. If the
reactions will be completed on a different day, prepare fresh
DNase dilutions.
7. The prepared DNA can be stored at −20 °C.
8. This step creates blunt ends by filling in the end or by chewing
away any overhanging DNA. Note the amount of T4 DNA
Ligase and T4 DNA Polymerase should be calculated based on
the amount of linear DNA.
9. Do not load more than 2.5 μg per lane (of the medium-sized
lanes). If possible, do not use UV light or ethidium bromide as
these can cause DNA damage.
10. Your primers should adhere to the following guidelines as
much as possible: (1) 40–60% GC, (2) at least 18 nucleotides
Engineering Protein Switches by Domain Insertion