PCR amplification of extracted DNA fragment

/PCR amplification of extracted DNA fragment
PCR amplification of extracted DNA fragment 2018-02-24T05:07:32+00:00

PCR amplification of extracted DNA fragment

PCR amplification of extracted DNA fragment

Introduction

The technique involves repeated rounds of in-vitro DNA synthesis by a thermo stable enzyme, such as Taq DNA polymerase which is isolated from bacteria called Thermus aquaticus. To perform a PCR reaction, PCR reaction mixture is prepared for which a small qwuatity of the target DNA ia added to an eppendorf with a buffer containing Taq DNA polymerase, oligonucteoide primers, the four deoxyribonucloetide of DNA (dNTPs), and the cofactor Mgcl2.

The PCR mixture is subjected to PCR cucles which consiste of following three steps

  1. Denaturation: During this step, DNA is denatured into single stranded DNA.DNA denaturation is the critical step in the DNA amplification reaction. The time specified for DNA depends on variuos factors like, nature of template, GC Content, secondary structure etc. For most amplifications, incubation time for DNA denaturation is 30 sec at 92o C to 95o C, with 94o C being standard choice.
  2. Primer Annealing: During this step, forward and reverse primers are annealed to their complementary sequences on either side of target sequence. The specified temperature is calculated empirically and is usually 5o C less than the Tm of the primers used. Extreme annealing temperature may result in non specific amplifications.
  3. Extension: During this step, annealed primers are extended by DNA polymerase to synthesize the compllementary DNA strands. Primer extension is usually carried out at a temperature of 72o C. Time used foir extension depends on thew length of target sequence.

Usually PCR is subjected to 30-40 cycles. As the amplification proceeds, the DNA sequences between the primers doubles after each cycle.

Requirements

Reagents

  1. Template DNA
  2. Mgcl2
  3. dNTPs
  4. Primers
  5. DNA Polymerase buffer
  6. Taq DNA Polymerase
  7. Sterile Water

Equipment

  1. Thermocycler

Procedure

  1. To prepare master mix and to minimize the possibility of pipetting errors, prepare a PCR master mix containing water, DNA buffer, MgCl2, dNTPs, primers and Taq DNA polymerase. Prepare suffiecient master mix fopr the number of reactions plus one into inbdiviiduals PCR tubes and then add DNA template.
  2. Gently vortex and briefly centrifuge all solutions after thawing.
  3. Place a thin walled PCR tube and add the following components for 50 µL reaction.
1OX assay Taq polymerase buffer 5 µL
dNTPs mix (2.5 mM each) 2 µL
Template DNA (100 ng/ µL) 2 µL
Forward primer (10 pM) 2 µL
Reverse primer (10 pM) 2 µL
Taq DNA polymerase (5U/µL) 1.25U (0.25 µL)
Sterile water 36.75 µL
Total reaction volume 50 µL

4. Gently mix and spin down the samples.
5. If using a thermal cycler that does not uses a heated lid, overlay the reaction mix with 25 µL of mineral oil.
6. Perform PCR using recommended thermal cycling conditions:

7. After the reaction is over, take out the reaction mix and check the samples in 1-1.5 % agarose gel for 1 to 2h at 90 volts. Run the sample along with marker and
locate the amplified product by comparing with those of the marker.
8. To visualize DNA keep the agarose gel on platform of UV transilluminator and switch on transmitted UV light to visualize EtBr stained DNA bands in the gel.
9 .Take photograph by a CCD camera attached with a computer.

Step Temperature C Time Number of cycles
Initial denaturation 94 1 – 3 min 1
Denaturation 94 30 sec 30 – 40
Annealing Tm – 5 30 sec
Extension 72 1 min/kb
Final extension 72 5-10 min 1
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