A Single-Tube Assembly of DNA Using the Transfer-PCR (TPCR) Platform - DNA Cloning and Assembly Methods - page 96

Stock
solution

Volume/20 μ L Final

concentration Component reaction

Forward primer 25 μ M 1 μ L 500 μ M

Reverse primer 25 μ M 1 μ L 500 μ M

μ

Master mix solution 2× 10 L 1×

Deionized, sterile water 8 μ L

2. Using a sterile toothpick select single colonies and transfer the
bacteria into the labeled 0.2 mL PCR tubes. Swirl well.
3. Using the same toothpick maintain each clone by inoculating liq-
uid or plate containing the appropriate antibiotic ( see Note 9 ).

4. Mix gently the 0.2 mL PCR tubes and spin briefl y.
5. Perform colony PCR reaction using the following parameters:

Step Temperature (°C) Time (min:s) Cycles

Denaturation 95 1:00 1

Denaturation 95 0:30 25

Annealing 60 1:00

Elongation 72 1:30

Elongation 72 6:00 1

Cooling 10 ∞

6. Analyze PCR reaction products on 1 % agarose gel to verify
that the DNA product obtained is of the correct size. Select
2-3 individual positive colonies ( see Note 12 ).
7. Inoculate positive clones into 10 mL LB medium supple-
mented with antibiotic in 50 mL conical tubes. Concentration
of the antibiotic depends on the type of the antibiotic used
(e.g., 30 μ g/mL of Kanamycin for cloning of IFN α 8 into

pET28-TevH).
8. Grow bacteria with shaking at 37 °C, for 16-20 h.
9. Harvest cells and extract DNA using a plasmid DNA purifi ca-
tion kit ( see Note 13 ).
10. Measure DNA concentration using a Nano-Drop
spectrophotometer.
11. Send the 2-3 positive selected clones for DNA sequencing
( see Note 14 ).
12. Transform plasmids from each positive clone to competent
BL21(DE3) E. coli ( see Note 15 ) cells and proceed with pro-
tein expression as described previously [ 13 , 14 ].