1.pET28a (+) vector and APEH synthetic gene were purchased from Eurofins.
2.Plate cells transformed with pET28a(+) vector on LB + kanamycin (30 mg/ml) and plate cells transformed with a plasmid containing the synthetic APEH gene on LB + ampicillin (100 mg/ml). Grow at 37 °C O/N in the incubator.
3.The day after, pick separately one colony of cells containing pET28a(+) in 5 ml of LB + kanamycin (30 mg/ml) and one colony of cells containing a plasmid with APEH gene in 5 ml of LB + ampicillin (100 mg/ml). Grow them at 37 °C O/N.
4.Then, extract the DNAs of pET28a(+) and of the vector containing APEH synthetic gene by using a Strataclone DNA midiprep kit. Measure DNA concentrations with a Nanodrop instrument. Control the quality of DNA by electrophoresis on agarose gel at 1%.
5.Digest 300 ng of both DNAs for 2 hours at 37 °C with 1 μl restriction enzymes Nde I and Hind III, which have compatible sticky ends for a ligation reaction. (Fig. 1A)
6.Check digestions on 1% agarose gel.
7.Cut from the gel the Nde I/Hind III DNA fragments from pET28a (+) and APEH plasmids.
8.Use an extraction kit to recover the DNA. (Fig. 1B)
9.Ligate the two NdeI/HindIII DNA fragments with T4 DNA ligase in the following reaction mixture: 50 ng of each DNA fragments, enzyme (400.000 U/ml), buffer 50 mM Tris-HCl, 10 mM MgCl2, 10 mM Dithiothreitol 1 mM ATP, pH 7.5, total volume 20 μL. The reaction is carried out at room temperature for 2 hours.
10.Heat-inactivate at 65°C for 10 minutes. Chill on ice.
11.Transform into competent cells.
12.Set up a PCR amplification of the APEH synthetic gene by using Pfu DNA polymerase and a pair of primers at the two ends of the region of interest, containing the two ligated species. Here we used T7 forward and T7 reverse primers (see Fig. 2). The sequences of T7 forward and T7 reverse primers were: 5’-TAATACGACTCACTATAGGG-3’ (Tm 53.2 °C) and 5’-GCTAGTTATTGCTCAGC-3’ (Tm 50.4 °C), respectively. Primers were purchased from Eurofins. Moreover, it is necessary to use a high-fidelity polymerase to preserve the sequence of the region to clone.
13.Check the PCR reaction on 1% agarose gel (Fig. 3)
14.Cut the gel fragment containing the band at 2532 bp, compatible with the length of HisTag-APEH gene.
15.Extract the DNA with a suitable kit (Promega).
16.Use 5 μl of ligase to transform the SoloPack cells.
17. On ice for 30 minutes.
18.Heat shock for 60 seconds at 42°C in a water bath.
19.Incubate 1 hour at 220 rpm at 37 °C.
20.Put 200 μl of cells onto a plate of LB + Kanamycine O/N at 37°C.
21.Denaturation phase at 95°C for 1 minute.
22.Annealing phase at 45°C for 1 minute.
23.Extension phase for 1.5 minutes (this step depends on the length of the base pairs involved).
24.Load 5 µl of each reaction into a 1% agarose gel, allowing a lane for an appropriate ladder, and perform electrophoresis.
25.Choose the positive clones.
26.Check the positivity of the DNA clone by sending it to a sequencing service (we used Eurofins Scientific).
|5||Null or partial digestions of DNAs||-Too high or too low concentrations of DNAs - NO compatible sticky ends for a ligation reaction -Restriction enzymes do not work||-Try different concentrations of DNAs and choose the best - Use only compatible sticky ends - Check the expire data and the concentration of the enzymes|
|22||No positive colonies||-Null or partial denaturation of DNA -Wrong set of extension phase||-Increase the time of denaturation phase from 1 minute to 3/5 minutes - Increase the time of annealing phase according to the length of the base pairs involved|