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1.-Confirm the sequence of
the cDNA to use by DNA sequencing.Confirm both: the sequence
corresponence to the Genbank sequence for this cDNA, and correspondence
of the translation
of this cDNA sequence to the expected protein sequence. |
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2.-Confirm the codon usage
of the cDNA in E.coli. |
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3.-Select a vector suitable
for your objectives: fusion protein, native protein, GST-purification,
Histidine Tag purification,... |
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4.-Select the restriction
sites to use for cloning the cDNA in the vector. Confirm that these
restriction sites are not present in the sequence of your cDNA, and are
unique in the sequence of the vector. Select preferably sites of
restriction enzymes with compatible buffers. Make atention to the conservation of
the correct reading frame. |
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5.-Design oligonucleotides
to add the selected restriction sites at the edges of the cDNA by PCR.
Make attention to the correct orientation of the CDNA sequence on the
vector to select the position of each restriction site, and confirm if
the correct reading frame will be respected. Assure the correct
digestion of the added restriction sites by adding some nucleotides at
the 5' edge of the sequence of each oligonucleotide before the sequence
coding for the restriction site.
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6.-Perform a PCR with these
primers on the cDNA. Purify the PCR product with the correct bp lenght. |
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7.-Digest separately the
vector and the purified PCR product with the selected restriction
enzymes. |
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8.-Purify and perform
ligation of the digested cDNA and Vector. |
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9.-Transform the resulting
plasmid on JM105 or DH5alpha E.coli. |
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10.-Confirm the obtained
clones by restriction analysis on Plasmidic DNA, preferably involving a
restriction site in the original cDNA sequence. Sequence and confirm at
least three of the clones positive for restriction analysis.Some
problems in the ligation could be detectable only by restriction
analysis, and not by DNA sequence. |
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11.-Transform the plasmidic
DNA of the selected clone to BL21 E.coli cells.Do not
sequence plasmides from BL21 cells, but from JM105 or DH5alpha.. As BL21 cells are protease
defective, often DNA minipreparations accumulate big amounts of
proteins and are not optimal for DNA sequencing. |
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12.-Test the ability to
synthesise protein of the resulting clone on 3ml cultures with/without
synthesis induction and clones with/without Plasmide.Confirm by PAGE-SDS
of culture protein extracts the Molecular Weight of the synthesised protein (the intense band present only in the
induced plasmid-containing clones).
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13.-Keep the selected BL21
E.coli clone frozen at -80C with glicerol. Use always the original
clone, do not recover the plasmid or the bacterial strain from protein
synthesis cultures for subsequent synthesis, as mutation of genomic or
plasmidic DNA during the synthesis can't be discarded. |
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