2X PCR Master Mix

PCR Amplification (2X Reaction Mix):

Cat No: 2X-MM-100RX

Introduction:

  • 2X Master PCR reaction mix is ready-to-use reaction mix containing proprietary DNA Polymerase, dNTPs and reaction buffer to efficiently amplify DNA templates by
  • To prepare for the final PCR, only primers and pure template DNA are needed to amplify targets DNA up to 3 k
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Description

Components Provided:

Product Vol.
1 2X Master PCR mix 2 X 1.25 ml
2 Molecular Biology Grade Water 3 ml

2 X Master PCR Mix: Comprises of proprietary Taq polymerase 2X Master PCR mix. It is ready-to-use mix for efficient amplification of DNA templates. PCR mix is made in nuclease-free water with optimal concentrations of Taq DNA Polymerase, dNTPs, MgCl2 and stabilizers.

PCR Protocol :                        

  1. Add 25 µl 2X Master PCR mix into a PCR
  2. Add 20 µl of nuclease free
  3. Add 2 µl of each primer from a working stock of 10 picomole/μl of each Final concentration of each primer should be 0.4 μM.
  4. Add 1 µl of your template DNA (10-20 nano grams)

Helpful Hints about primers concentration:

a).Forward and Reverse primers are generally 20–40 nucleotides in length with GC content of 45 to 65%. The final concentration of each primer in a reaction varies from 0.05–1 μM. If you are not sure of the right primers concentrations for a PCR reaction, We recommend with a final concentration for 0.4 µM of each primer.

  • If you have a primer suspended in sterile water at a concentration of 100 picomoles/μl, dilute it 10 fold to final concentration of 10 picomoles/μl (10 μM) for each working Add 2 μl of each primer to 50 μl of PCR volume.
  • If you add 2 µl of a 10 μM stock into a 50 μl reaction, then the concentration of your each primer in the 50 μl reaction would be 2/50th of 10 μM, or 4 μM (400 nM).
  • If your PCR primer is supplied as 40 nmol per tube dried, suspend it in 400 μl of sterile water, which will be equivalent to 100 picomoles/μl (100 μM)
  1. Vortex samples for few seconds or mix the contents by pipetting up and down with a
  2. If PCR machine does not have a heated lid, add two drops of mineral oil into each
  3. Use following conditions for a thermocycler (if your fragment size is about 1 KB):

Denaturing Step: 95 °C for 5 minutes Amplification Steps (35 steps)

  1. 95 °C for 1 minute (Denaturing step)
  2. 60 °C for 1 minute (annealing step)
  3. 72 °C for 1 minute (extension step)

Final extension Step:

  1. 70 °C for 5 minutes (final extension)

Helpful Hints about extension time: An extension time of 1 minute per Kb is recommended. Extension time from of 20 seconds ~ (300bp) to 5 minutes ~ (5 Kb) can be used depending upon the size of your fragment.

Troubleshooting                         

Problem Possible Cause Suggestions
  

 

 

 

 

 

 

 

 

No or low PCR products

Not Sufficient Template •      Increase concentration of DNA template
Target sequence missing in the 

template

•      Re-extract the DNA template from the source.
Incorrect Primers design •      Ensure that the primers are specific and are 

complementary to the target of interest.

Primers Concentration too low •      Increase the concentration of primers 

•      Optimize primers concentration (0.1-1 µM)

Primers quality •     Reconstitute fresh primers, aliquot primers after reconstitution and store properly.
Insufficient number of PCR 

cycles

•      Increase the number of cycles (generally 25-35 

cycles).

•      If need be extend the number of cycles to 40.

  

 

 

Denaturation, annealing and extension are not optimal

•      Optimize the DNA denaturation temperature 

and time.

•     Optimize the annealing temperature time. The optimal annealing temperature is usually 3-5 °C below the lowest primer Tm.

•     Optimize the extension temperature based on the amplicon length. Optimal extension temperature: 72°C.

•     Reduce the extension temperature to 68°C for amplification of long targets.

Inhibitor (s) present in the reaction mix •      Purify templates by alcohol precipitation or by 

using clean up kit.

•      Optimize the sample volume.

Incorrect thermocycler program •      Verify program for temperatures and times.
Multiple band 

/non-specific PCR Products

 

 

 

 

 

 

 

 

 

 

 

Smear PCR products

Non-optimal template concentration •      Use 1 pg-10 ng of plasmid DNA/50 µl reaction. 

•      Use 1 pg-10 ng of genomic DNA/50 µl reaction

•      Isolate new template

Contaminated template
Poor primer design •      Explore literature for proper primer design. 

•      Verify the complementarily of both primers

•      Increase length of primers to 24 bases.

•      Avoid GC-rich 3’ ends

•      Use lower concentration of primers.

Too high concentration of 

primers

•      Primer concentration should stay in the range of 

0.05-1 µM in the reaction.

•      Check GC content of the primers

GC content in the primers in 

no appropriate

•      Run gradient temperature PCR 

•      Use freshly prepared new reaction mix.

Contaminated reaction 

mixture components

•      Wear gloves and use sterile and clean filter 

pipette tips during setting up of the reaction.

•      Recalculate primer Tm values.

Too low or incorrect annealing 

temperature

•      Increase annealing temperature, but 

intermittently.

•      Prefer using a hot start DNA polymerase

Premature replication •      Change the primers.
Template degraded, primers degraded or both template and primers are contaminated •      Use fresh non-degraded template DNA. 

•     Change PCR tubes and try different thermocycler.

•      Use PCR grade sterile water.

•     Check the quality of DNA template by electrophoresis.

•      Check 260/280 ratio of DNA template.

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