3. Na 3 VO 4 should be activated for maximal inhibition of protein
phosphotyrosyl-phosphatases as follows:
(a) Prepare a 200 mM solution of Na 3 VO 4 .
(b) Adjust the pH to 10 using either 1 N NaOH or 1 N HCl.
At pH 10 the solution will be yellow.
(c) Boil the solution until it turns colorless.
(d) Cool to room temperature.
(e) Readjust the pH to 10 and repeat steps 3c and 3d until
the solution remains colorless and the pH stabilizes at 10.
(f) Store the activated Na 3 VO 4 in aliquots at À20
C.
4. The pGEM-AGO plasmids are mutagenized by the Quickchange method wherein a pair of complementary mutagenic
primers are used to amplify the entire plasmid in a PCR reaction
using a high-fidelity DNA polymerase such as Pfu polymerase
or Phusion. Mutagenic primers contain the desired mutation
and are complementary to the template DNA around the
mutation site (approximately 15 nt up- and downstream of
the mutation). If possible choose triplets, which creates a new
or eliminates an existing restriction site; thereby the mutant
plasmid can be identified by restriction digestion. An important
step of the method is the elimination of the original
non-mutagenized template. This is performed by DpnI restriction digestion. DpnI preferentially degrades the methylated
template plasmid molecules (produced by the bacterial host),
while leaves the unmethylated plasmid molecules (produced
during the mutagenic PCR) intact. With this simple procedure
significant enrichment of the mutagenized plasmids can be
achieved.
5. The annealing temperature used in PCR mutagenesis does not
correspond exactly to the calculated Tm values of the mutagenic primers. Annealing temperature between 64 and 54
C
should be used. Start at higher temperatures. If the number of
growing E. coli colonies after mutagenesis is low (indicating
that the procedure of PCR mutagenesis was not successful)
decrease the annealing temperature gradually.
6. Good-quality chemically competent E. coli can be prepared
following the Inoue protocol [16]. High-efficiency competent
E. coli cells are also available from a number of commercial
sources.
7. Mutagenesis has to be confirmed by sequencing. The pGEM-T
easy vector contains SP6 and T7 primer sites flanking the polylinker. These primers can be used for sequencing. The entire
AGO insert has to be sequenced to confirm that only the
desired mutation is introduced into the ORF. Alternatively, a
shorter DNA segment around the introduced mutation can be
An In Vivo Reporter System for RNA Silencing
447
phosphotyrosyl-phosphatases as follows:
(a) Prepare a 200 mM solution of Na 3 VO 4 .
(b) Adjust the pH to 10 using either 1 N NaOH or 1 N HCl.
At pH 10 the solution will be yellow.
(c) Boil the solution until it turns colorless.
(d) Cool to room temperature.
(e) Readjust the pH to 10 and repeat steps 3c and 3d until
the solution remains colorless and the pH stabilizes at 10.
(f) Store the activated Na 3 VO 4 in aliquots at À20
C.
4. The pGEM-AGO plasmids are mutagenized by the Quickchange method wherein a pair of complementary mutagenic
primers are used to amplify the entire plasmid in a PCR reaction
using a high-fidelity DNA polymerase such as Pfu polymerase
or Phusion. Mutagenic primers contain the desired mutation
and are complementary to the template DNA around the
mutation site (approximately 15 nt up- and downstream of
the mutation). If possible choose triplets, which creates a new
or eliminates an existing restriction site; thereby the mutant
plasmid can be identified by restriction digestion. An important
step of the method is the elimination of the original
non-mutagenized template. This is performed by DpnI restriction digestion. DpnI preferentially degrades the methylated
template plasmid molecules (produced by the bacterial host),
while leaves the unmethylated plasmid molecules (produced
during the mutagenic PCR) intact. With this simple procedure
significant enrichment of the mutagenized plasmids can be
achieved.
5. The annealing temperature used in PCR mutagenesis does not
correspond exactly to the calculated Tm values of the mutagenic primers. Annealing temperature between 64 and 54
C
should be used. Start at higher temperatures. If the number of
growing E. coli colonies after mutagenesis is low (indicating
that the procedure of PCR mutagenesis was not successful)
decrease the annealing temperature gradually.
6. Good-quality chemically competent E. coli can be prepared
following the Inoue protocol [16]. High-efficiency competent
E. coli cells are also available from a number of commercial
sources.
7. Mutagenesis has to be confirmed by sequencing. The pGEM-T
easy vector contains SP6 and T7 primer sites flanking the polylinker. These primers can be used for sequencing. The entire
AGO insert has to be sequenced to confirm that only the
desired mutation is introduced into the ORF. Alternatively, a
shorter DNA segment around the introduced mutation can be
An In Vivo Reporter System for RNA Silencing
447