Question: What goes in a PCR re.............. Edit
Answer: PCR reaction tube contains:
i. Template DNA: It is the DNA of interest which one wants to amplify. Higher amount
of template can lead to non-specific amplification. Generally, 1ng amount of plasmid DNA
and 1µg of genomic DNA is used.
ii. Primers: Two primers, one forward and one reverse primer usually ranging between 16-30
bases are required. Primer length less than 10 bases may result in non-specific annealing.
Primers should be free from secondary structures. Optimal concentration of primers is
between 0.1-1.0 µM. Primers are specific sequence of short stretch of DNA which can bind
specific site of target DNA to initiate the replication via Taq Polyemerase.
iii. Deoxynucleotide-tri-phosphate: The final concentration of each dNTP in a standard
amplification reaction is 200 µM. It acts a raw material to synthesize new strands of DNA of
iv. Taq DNA Polymerase Buffer: The 10X assay buffer contains 100 mM Tris-Cl (pH-
9.0), 500 mM MgCl2 and 0.1% w/v gelatin. Mg2+ is an essential cofactor. Low concentration
of Mg2+ will result no amplification and high concentration may lead to the production of
nonspecific products. Buffer mainly keeps a constant pH for optimum enzymatic activity and
also stabilize the structure of product.
v. Taq DNA Polymerase: It is a 94 KD thermo stable DNA polymerase. Its optimal
temperature is 720C. It lacks 3´- 5´ exonuclease activity but has 5´- 3´ polymerase activity. It
is special DNA polymerase which can polymerize dNTP at relatively high temperature
suitable for PCR.
PCR Master Mix:
It is a premixed, ready-to-use solution containing Taq DNA polymerase, dNTPs, MgCl2
and reaction buffers at optimal concentrations for efficient amplification of DNA templates by PCR.
i. Supplied DNA sample (1ng/ µl) 1µl
ii. 10µM Primer (forward & reverse):- 1µl 2 µl
iii. 10X Buffer:- 2µl 4 µl
iv. 10mM dNTPs:- 0.4µl 0.8 µl
v. Taq DNA Polymerase (3units/µl):- 0.3µl 0.6 µl
vi. H2O:- 15.3µl 30.6 µl
20.0µl 38 µl (Mastermix for two reactions)
Negative control, a reaction without any template must be performed in parallel. The band
of desired product must be absent in negative control. For a set of reactions, preparation of mastermix
including all components, except template DNA is recommended. Edit
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