How To Clone:
Part I: Collecting Genomic DNA
1. After purifying to collect bacteria, cell lysis is performed by using EDTA and SDS to
disintegrate the cell wall and plasma membrane.
2. The cell extract is centrifuged to remove all debris shy of DNA, RNA and proteins.
3. This new supernatant is mixed with the solvent phenol in a 1:1 ratio and again the extract
is spun in a centrifuge to separate the aqueous nucleic acid layer.
4. Absolute ethanol (100%) precipitation and addition of sodium acetate salt (at -20°C)
dissolves the now neutralised DNA and RNA out of its aqueous solution via
centrifugation.
5. The absolute ethanol is removed and 70% ethanol is added to the pellet to dissociate off
the sodium ions and nally dissolve pure DNA/RNA into the ethanol.
Part II: Collecting Plasmids
1. Alkaline denaturation is performed (pH12.5) on some organism that has the plasmids we
want to extract where after puri cation of nucleic acids, the denaturation step breaks
down dsDNA but has no effect on supercoiled plasmids.
2. The pH is re-established to pH7 where linear genomic DNA and proteins precipitates off
(after centrifugation) into a tangled mass while the plasmids remain in the supernatant.
Part III: Cutting Up Genes and Vectors
1. Type 2 restriction enzymes that cut up nucleic acids at speci c sequences are used to
isolate and cleave genes and vectors respectively.
2. Sticky ends are generated in plasmids and on the two ends of a gene cut out to eventually
glue these together (intermolecular association).
3. For Plasmids: The restriction enzyme used should only cut at one place otherwise the
vector is cleaved into two or more fragments and that would be useless.
4. For Genes: The restriction enzyme used on an entire genome will have hundreds to
thousands of cleaving sites as an entire genetic complement will have many repeats of
the RE-speci c sequence. Tetra-nucleotide REs will cut up fragments that are 256bp long,
while hexa-nucleotide REs will cut fragments of length 4096bp long, and since most genes
are ±1000bp long and thus either you can use a tetra-nucleotide RE and have it partially
digest the DNA for a short amount of time, or more reliably use a hexa-nucleotide RE that
also in a partial digest is far more likely to excise a gene of interest.
Part IV: Joining Genes and Vectors
1. T4 ligase is the enzyme used to anneal the complementary sticky ends of the plasmid and
gene and requires ATP as a co-factor.
2. Ligating sticky ends is far easier than ligating blunt ends, and with discontinuous sticky
ends being annealed via T4 ligase at their shared nicks to o cially join the two together.
3. These reactions are carried at at high concentrations, where there is a ratio of 3:1 of the
insert gene to the vector DNA to prevent vector DNA from re-annealing (although this is
bound to happen regardless).
Part V: How to Anneal
1. The nick in the dsDNA of the combined vector and gene discontinuous sticky end is
identi ed by T4 ligase via a lysine residue which de-phosphorylates ATP and then binds to
the subsequent AMP.
2. The enzyme-AMP complex phosphorylates the 5’ phosphate end on the one strand where
the DNA bound phosphate attaches to the AMP which dissociates off of the T4 ligase.
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Part I: Collecting Genomic DNA
1. After purifying to collect bacteria, cell lysis is performed by using EDTA and SDS to
disintegrate the cell wall and plasma membrane.
2. The cell extract is centrifuged to remove all debris shy of DNA, RNA and proteins.
3. This new supernatant is mixed with the solvent phenol in a 1:1 ratio and again the extract
is spun in a centrifuge to separate the aqueous nucleic acid layer.
4. Absolute ethanol (100%) precipitation and addition of sodium acetate salt (at -20°C)
dissolves the now neutralised DNA and RNA out of its aqueous solution via
centrifugation.
5. The absolute ethanol is removed and 70% ethanol is added to the pellet to dissociate off
the sodium ions and nally dissolve pure DNA/RNA into the ethanol.
Part II: Collecting Plasmids
1. Alkaline denaturation is performed (pH12.5) on some organism that has the plasmids we
want to extract where after puri cation of nucleic acids, the denaturation step breaks
down dsDNA but has no effect on supercoiled plasmids.
2. The pH is re-established to pH7 where linear genomic DNA and proteins precipitates off
(after centrifugation) into a tangled mass while the plasmids remain in the supernatant.
Part III: Cutting Up Genes and Vectors
1. Type 2 restriction enzymes that cut up nucleic acids at speci c sequences are used to
isolate and cleave genes and vectors respectively.
2. Sticky ends are generated in plasmids and on the two ends of a gene cut out to eventually
glue these together (intermolecular association).
3. For Plasmids: The restriction enzyme used should only cut at one place otherwise the
vector is cleaved into two or more fragments and that would be useless.
4. For Genes: The restriction enzyme used on an entire genome will have hundreds to
thousands of cleaving sites as an entire genetic complement will have many repeats of
the RE-speci c sequence. Tetra-nucleotide REs will cut up fragments that are 256bp long,
while hexa-nucleotide REs will cut fragments of length 4096bp long, and since most genes
are ±1000bp long and thus either you can use a tetra-nucleotide RE and have it partially
digest the DNA for a short amount of time, or more reliably use a hexa-nucleotide RE that
also in a partial digest is far more likely to excise a gene of interest.
Part IV: Joining Genes and Vectors
1. T4 ligase is the enzyme used to anneal the complementary sticky ends of the plasmid and
gene and requires ATP as a co-factor.
2. Ligating sticky ends is far easier than ligating blunt ends, and with discontinuous sticky
ends being annealed via T4 ligase at their shared nicks to o cially join the two together.
3. These reactions are carried at at high concentrations, where there is a ratio of 3:1 of the
insert gene to the vector DNA to prevent vector DNA from re-annealing (although this is
bound to happen regardless).
Part V: How to Anneal
1. The nick in the dsDNA of the combined vector and gene discontinuous sticky end is
identi ed by T4 ligase via a lysine residue which de-phosphorylates ATP and then binds to
the subsequent AMP.
2. The enzyme-AMP complex phosphorylates the 5’ phosphate end on the one strand where
the DNA bound phosphate attaches to the AMP which dissociates off of the T4 ligase.
fi fi fi fi ffifi
