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RTPCR Droso

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18 views2 pages

RTPCR Droso

Uploaded by

aryanyadav72003
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Download as DOCX, PDF, TXT or read online on Scribd
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Protocol for PCR master mix:

To perform several parallel reactions, prepare a master mix containing water, buffer, dNTPs,
primers, Taq DNA polymerase in a single tube, which can then be aliquoted into individual
tubes. This method of setting reactions minimizes the possibility of pipetting errors and saves
time by reducing the number of reagent transfers. Template DNA solution is added after
aliquoting into tubes.

1. Determine the volume of reaction to be amplified (30 µl).


2. Gently vortex and briefly centrifuge all solutions after thawing.
3. In a separate microtube, mix the reagents (based on the calculated volume) by first
putting the water followed by buffer, primers, dNTPs and Taq.
4. Carefully label the tubes and dispense the PCR master mix into each tube (200µl PCR
tube).
5. Add DNA (Template) to each tube.
6. Place the tube in the thermal cycler programmed with the desired temperature and
time of amplification.

Final concentration 30µl reaction

Sterile distilled water 21.7

10X buffer with Mg2+ 1X 3 µl

10mM dNTPs 0.2mM of each 1 µl

Primer F 10 pmol 1 µl

Primer R 10 pmol 1 µl

Taq polymerase 1 unit 0.3 µl

PCR STEPS:

1. Denaturation step: This step is the first regular cycling event and consists of heating the
reaction to 94-98°C for 20-30 seconds. It causes melting of DNA template and primers by
disrupting the hydrogen bonds between complementary bases of the DNA strands, yielding
single strands of DNA.
2. Annealing step: The reaction temperature is lowered to 50-65°C for 20-40 seconds allowing
annealing of the primers to the single-stranded DNA template. Typically the annealing
temperature is about 3-5 degrees Celsius below the Tm of the primers used. Stable DNA-
DNA hydrogen bonds are only formed when the primer sequence very closely matches the
template sequence. The polymerase binds to the primer-template hybrid and begins DNA
synthesis.
3. Extension/elongation step: The temperature at this step depends on the DNA polymerase
used; Taq polymerase has its optimum activity temperature at 72-80°C, and commonly a
temperature of 72°C is used with this enzyme. At this step the DNA polymerase synthesizes a
new DNA strand complementary to the DNA template strand by adding dNTP's that are
complementary to the template in 5' to 3' direction, condensing the 5'-phosphate group of the
dNTPs with the 3'-hydroxyl group at the end of the nascent (extending) DNA strand. The
extension time depends both on the DNA polymerase used and on the length of the DNA
fragment to be amplified. As a rule-of-thumb, at its optimum temperature, the DNA
polymerase will polymerize a thousand bases in one minute.
NOTE : Final elongation: This single step is occasionally performed at a temperature of 70-74°C for 5-
15 minutes after the last PCR cycle to ensure that any remaining single-stranded DNA is fully extended.

Final hold: This step at 4°C for an indefinite time may be employed for short-term storage of the
reaction.

Agarose gel electrophoresis of PCR product:

The dye used for the visualization of amplified PCR bands in agarose gel electrophoresis is
ethidium bromide (EtBr). It has the unique property of fluorescing under UV light when
intercalated with PCR product. By running PCR product through an EtBr-incorporated gel
and exposing it to UV light, distinct band of PCR product visible. Loading buffers are added
with the PCR product in order to keep track of the position of PCR product during
electrophoresis and to sediment it in the gel well. Xylene cyanol and Bromophenol blue are
generally used.

Result:

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