WB ICC/IHC
2.12 Expression plasmid construction and cloning
2.12.1 General DNA construct amplification
All DNA constructs were amplified using 1.25 units of Pfu DNA polymerase and the corresponding buffer with 10 pmol forward primer, 10 pmol reverse primer and 10 mM dNTPs (each) in a thermocycler. The optimal cycling parameters were adjusted according to the melting temperature of the primer pair and the amplicon size. The annealing temperature was set at 5°C below the melting temperature of the primer pair and the elongation time at 72°C was calculated according to the polymerase, 1 min for each 500 basepairs of DNA to be amplified. The cycles were repeated 30 times.
PCR Program
1. Denaturation 95°C 3 min
2. Denaturation 95°C 30 sec 3. Annealing x °C 30 sec 4. Elongation 72°C x min
go to step 2 for another 29 cycles 5. Final elongation 72°C 5 min
The size and the quality of the PCR product was determined with agarose gel electrophoresis. As marker a 1 kb or 100 bp ladder was used depending on the size of the PCR product. PCR fragments were cut out and extracted using the Nucleo Spin® Extract II Kit following the manual.
Materials and Methods
2.12.2 Cloning
Cleaned PCR products and the respective target expression vectors were digested in a minimal volume of 50 μl with the corresponding restriction enzymes for 1‐2 hr at 37°C.
Expression vectors were also digested subsequently with alkaline phosphatase, which catalyzes the removal of 5´ phosphate groups of DNA to prevent self‐ligation. PCR products were directly purified using Nucleo Spin® Extract II Kit, whereas expression vectors were analyzed with agarose gel electrophoresis on a 1% agarose gel to confirm efficient linearization. Only linearized vector was cut out and cleaned with the Nucleo Spin® Extract II kit. To ligate 16 µl of digested PCR product, 1 µl of digested target expression vector, 2 µl of ligation buffer and 1 µl T4 DNA ligase were mixed together and ligated either 1 hr at RT or overnight at 4°C. Entire ligation reaction was transformed into 100 μl of chemo‐
competent DH5α E. coli cells, incubated on ice for 20 min, heat shocked at 42°C for 1 min and then replaced on ice for 1 min. Afterwards, 1 ml of prewarmed 2xYT media without antibiotics was added and cells were incubated for 1 hr at 37°C. Cells were spun down at 4000 rpm for 3 min and plated on a 2xYT agar plate with 50 μg/ml ampicilline or kanamycin depending on the resistance gene carried by the vector for selection. Plates were incubated overnight at 37°C.
For verification of positives clones, colonies were picked with an autoclaved pipett tip and transferred into 2 ml of 2xYT media with the appropriate antibiotics. These mini‐
cultures were incubated overnight at 37°C. Then, plasmid DNA was isolated with an alkaline lysis protocol and subjected to restriction digest to detect successful ligation. The Nucleo Spin® Plasmid Quick Pure was used to purify DNA from confirmed positive clones followed by sequencing analysis by the AGCTLab at the MPI of Experimental Medicine. For alkaline lysis, cell suspension was transferred into a 2 ml tube and spun down for 3 min at 13000 rpm. Pellet was resuspended in 200 µl of GTE buffer with freshly added RNase (5 µl of 10 mg/µl stock solution) and incubated at RT for 5 min. 400 µl of freshly prepared bacteria lysis buffer was added and mixed gently by inverting the tube. Sample was placed on ice for 5 min. Then, 300 µl of ice cold neutralization buffer (3M potassium acetate) was added, mixed gently and incubated on ice for 5 min. Sample was centrifuged for 5 min at 13000 rpm. Supernatant was transferred into a new tube and 540 µl of isopropanol was added. By mixing the two solutions the DNA was precipitated. Sample was incubated for 15 min at RT and then centrifuged at max speed for 2 min. Pellet was washed once with 70%
ethanol, air‐dried and resuspended in 50 µl of H2O. 5 µl of DNA was used for restriction
Materials and Methods
digestion with appropriate enzymes and analyzed by gel electrophoresis on a 1% agarose gel.
For transfection of plasmid DNA into heterologous cells or primary neurons, plasmid DNA was amplified in chemo‐competent DH5α E. coli cells in 50 ml or larger volumes of 2xYT medium with the appropriate antibiotics overnight in a 37°C shaker.
Plasmid DNA was purified with the Nucleo Bond® Xtra Midi EF kit according to manufacturer’s instructions.
2.12.3 Sitedirected mutagenesis of plasmids
Site‐directed mutagenesis was used to mutate the RNAi targeting sequence in pCMV‐myc‐RNF157 and pCMV‐myc‐Fe65 that renders these encoded molecules resistant to RNAi‐mediated degradation. The primer pairs 22180/ 22181 with 6 silent mutations for RNF157 and 23636/23637 with 4 silent mutation for Fe65 in the sequence were designed according to the stratagene/Agilent technology manual using the provided formula:
Tm = 81.5 + 0.41(%GC) (675/N) % mismatch
N is the primer length in bases and values for %GC and %mismatch are integral numbers.
The protocol was then modified in the laboratory. 15 pmol primers were used in a PCR reaction using the Pfu DNA polymerase and the respective vector as template (10 ng). The following cycling parameters were used:
Sitedirected mutagenesis PCR
Denaturation 3 min
Denaturation 30 sec Annealing (55°) 30 sec
Elongation 1 min/500 bp
17 cycles
Final Elongation 5 min
Sample was then incubated on ice for 2 min, digested with DpnI for 1 hr at 37°C and chilled on ice for 10 min. 1 µl of the digested PCR product was transformed into chemo‐competent DH5α E. coli cells, plated on a 2xYT agar plate containing the appropriate antibiotics and incubated overnight at 37°C. Successful site‐directed mutagenesis was confirmed by sequencing.
Materials and Methods
2.12.4 shRNA design and plasmid construction
The RNAi technology used in this study is based on the expression of short hairpin RNAs from a DNA template under the control of the RNA polymerase III U6 promoter. The oligonucleotide primers were designed manually in accordance with the following rules.
The 5´ sense primer should start either with AA, AG or GG followed by 18 nucleotides of the target DNA sequence and should have a GC content of 45‐60%. This sequence has to be specific for the target gene, should show no homologies to any other genes and contain not more than three adenosines in a row. Additionally, the targeting region should be identical between mouse, rat and human. The target sequence is followed by a modified Brummelkamp loop (TTCAAGCTT, with a unique HpaI restriction site) and the antiparallel target sequence with addition of CTTTTG (= Stop signal for the RNA polymerase III). The 3´
antisense primer is complementary to the sense primer with addition of TTAA at the end. 2 µl of each primer (50 pmol/µl) was resuspended in 46 µl of annealing buffer, incubated for 4 min at 95°C, 10 min at 72°C and slowly cooled down (0.1°C/sec) to RT. Then, 20 µl of the annealed oligonucleotides were phosphorylated in a kinase reaction for 30 min at 37°C. In parallel to primer preparation, the pBluescript‐U6 vector was digested with ApaI, blunted with T4 DNA polymerase, digested with EcoRI and dephosphorylated with calf intestinal phosphatase (CIP). Finally, 3‐5 µl of phosphorylated oligonucleotides and 1 µl of the pBluescript‐U6 vector were used for ligation overnight at 4°C and transformed into chemo‐
competent DH5α E. coli cells. Verification of positive clones was done by digestion with HpaI and analyzed by gel electrophoresis on a 1% agarose gel.