Aliquots of 2 µl of ligation product were incubated at room temperature for 5 min with 3 µl of StrataClone Cloning Buffer and 1 µl of StrataClone vector Mix.
The 10 μl PCR reaction included 0.8 μl glycerol stock of the clone, 1X PCR buffer, 0.15 mM of each dNTP, 1.8 mM MgCl2, 0.15 μM of reverse and forward primers, and 0.8 units of Taq polymerase.
Concentration was measured using a NanoDrop, and up to 30 μg RNA per sample was treated with DNase I in 100 μl reaction volumes for 30 min at 37° (reaction mix: 4 U Cloned DNase I TaKaRa 2220A, 80 U Promega Recombinant RNasin N2515, in 1× TaKaRa Cloned DNase I Buffer II).
For western analysis, 10 μg aliquots of protein extract from control and transgenic clones in Laemmli buffer were separated on 4-20% Tris Glycine polyacrylamide gel (PAGEr® Gold Precast Gel, Biorad) according to the manufacturer's instructions.
Subsequently, 100 μl of the biotinylated detection antibody solution (250 μg l−1 anti-KLK6 monoclonal antibody clone E24 in assay buffer) was added to each well.
Coating of Maxisorp flat-bottomed 96-well microtiter plates (Dako, Glostrup, Denmark) was done overnight at 4°C adding 100 μl of 2.5 μg ml−1 anti-human TFF3 M01 clone 3D9 in carbonate/bicarbonate buffer (Na2CO3 0.0015 M, NaHCO3 0.035 M) pH 9.6.
PCR fragments were analyzed by electrophoresis in a 1% agarose gel (1x TAE buffer) and cloned into pJET1.2/blunt vector using the CloneJET PCR Cloning Kit (Fermentas).
For the control plates, we added 4 × 107 cells in 400 μl of KK2 buffer for each clone.
The 3.3-kb TaEPSPS-7A1 clone was successfully recovered using buffer D and 5 % DMSO, and sequenced in both the forward and reverse directions (Table 1).
Triplicate wells for each clone were harvested in lysis buffer (Promega, No. 397A) at 24 h intervals after PBS wash (x3).
