Biology Reference
In-Depth Information
5. We recommend using a total volume of 20
L for the ligase
reaction. Use 1 U ligase for each ligation. Mix all components
by pipetting.
6. Incubate for 1 h at room temperature or at 16 °C overnight
(
see
Note 9
).
7. Remove the proteins from the solution by phenol-chloroform
extraction: Bring the ligation mixture up to 200
μ
L with H
2
O,
then add an equal volume of PC8 to the DNA containing
reaction mixture and vortex (
see
Note 10
). Then separate the
aqueous phase which contains the DNA from the organic
phase by centrifugation at 16,000 ×
g
for 5 min. Lastly, transfer
the aqueous phase with care into a fresh (labeled) reaction
tube.
8. Ethanol-precipitate the nucleic acid by adding 0.1 volume of
3 M sodium acetate, pH 5.2 and 2
μ
L of glycogen to the aque-
ous phase, vortex, then add 2 volumes of absolute ethanol and
vortex again. Recover the precipitated DNA by centrifugation
at 16,000 ×
g
for 5 min. Then, remove the ethanol with care
(
see
Note 11
) and wash the pellet three times with 70 % etha-
nol. Finally, dry the pellet at room temperature or 37 °C and
dissolve it in 10
μ
μ
L H
2
O.
5
Transformation of Electroporation-Competent
E
.
coli
1. Thaw electroporation-competent bacteria cells on ice (we rec-
ommend the use of bacteria with a transformation effi ciency of
at least 1 × 10
8
cfu/
μ
g).
2. Chill 2-3
L of the ligation mixture in a 1.5 mL reaction tube.
3. Add competent cells to the DNA. Mix gently by pipetting or
fl icking the tube 4-5 times to mix the cells and DNA. Do not
vortex.
4. Transfer the DNA-cell mixture into a chilled electroporation
cuvette (0.1 cm gap).
5. Put the dry cuvette into the sample chamber of an electropora-
tor and pulse once at 1,800 V.
6. Remove the cuvette from the sample chamber and add 1 mL
of LB-medium immediately (
see
Note 12
).
7. Transfer the cell suspension into a 2 mL reaction tube.
8. Incubate the cell suspension for 45-60 min at 37 °C while
shaking at 190-220 rpm.
9. Plate aliquots of the bacteria suspension (
see
Note 13
) on
LB-agar plates supplemented with antibiotics.
10. Incubate plates at 37 °C overnight.
μ
