Application of
biomolecular tools
for cloning of a foreign gene
PCR amplification
●
DNA sample – EQ lysosyme.
●
“Home-made” Taq polymerase + buffer.
●
PCR cycle.
Denaturation – 95°C, 30 seconds.
Annealing – 60°C, 1 minute.
Elongation – 72°C, 2 minutes.
25 cycles were run.
Restriction digest of vector
●
pET-28-blue T-vector (500 ng).
●
NcoI + HindIII.
Incubation at 37°C, one hour.
Inactivation by purification with
QIAgen minelute PCR purification kit.
Agarose gel electrophoresis
●
●
5 μl PCR product
(non-purified), well 4.
2,5 μl cleaved
vector, well 3.
●
Lookin' good!
We have clearly visible
bands at the correct
molecular weight.
Purification and restriction
digest of PCR product
●
Purified with QIAgen Minelute PCR
purification kit.
●
Restriction digest – NcoI + HindIII.
●
Purified again as above.
Modification of vector
●
1 μl alkaline phosphatase (10ng/ml),
2 μl buffer, 8 μl vector, 9 μl H2O, incubated at
37°C for one hour.
This removes a phosphate group from the 5' end, to prevent self-ligation of the vector.
●
The vector was again purified with the
QIAgen kit.
Ligation of vector
and PCR product
●
●
7 μl vector (10 ng/ml), 3 μl PCR product
(around three-fold molar excess of PCR
product), 1 μl Quick T4 DNA ligase, 10 μl 2x
Quick ligation buffer.
Incubated at room temperature for 15 minutes.
Transformation
●
50 μl Nova Blue supercompetent cells,
21 μl ligated vector, incubated on ice for 30
minutes.
●
●
●
Heat shock on heating block, 42°C, 45 seconds,
then placed on ice for two minutes.
Add 200 μl pre-heated SOC culture medium,
incubate at 37°C for one hour.
Spread 200 μl on an agar plate, incubate at
37°C for at least 16 hours.
Preparation for analysis
●
●
●
Colonies acquired ~ 370.
Four colonies were picked and added to
separate PCR tubes. All PCR components
except Taq polymerase was added and
then heated at 95°C for five minutes.
Taq polymerase was added, and a PCR
cycle was run 25 times at
95°C, 30 seconds → 60°C, 30 seconds → 72°C, one minute.
Results
●
●
Each tube was run on an agarose gel
electrophoresis. We received no results at
all. Lookin' bad! (missing picture of the agarose gel)
The probable reason for our negative results are a faulty ligation
reaction, possibly because one of the restriction enzymes was not
working properly. This will have resulted in a self-ligation of the
vector/PCR product. This could also be the reason why we acquired
a lot of colonies. Another possible reason could be that we were
simply unlucky when picking our colonies. We might as well have
lost some product during the purification steps (while eluating the
product from the minelute column).
Authors
Sofia Edström
Johan Fogelholm
Marcus Fransson
Madeleine Gabrielson
Jenny Sahlin
Karoline Sverkström
… och krya på dig Lasse! =)