Description

Recommended PCR Condition

2X Master Mix Prepration

* Optimal amount of DNA template depends on the source and quality of DNA. TAQ DNA Polymerase: 1.25 unit (0.25 μl) per 50 μl reaction is recommended. Please Use 2~2.5 unit in case target size is > 3kb.

10X Buffer Includes : 15mM MgCl2

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. b). 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 primer. 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 prime 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)

5. Vortex samples for few seconds or mix the contents by pipetting up and down with a
6. If PCR machine does not have a heated lid, add two drops of mineral oil into each
7. 1. 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 3 minutes ~ (3 Kb) can be used depending upon the size of your fragment.

8. 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 0C below the lowest primer Tm. •     Optimize the extension temperature based on the amplicon length. Optimal extension temperature: 72 0C. •     Reduce the extension temperature to 68 0C 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 Use 1 pg-10 ng of genomic DNA/50 µl reaction
   	Contaminated template	•      Isolate new 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
   	Too high concentration of primers	•      Use lower concentration of primers. •     Primer concentration should stay in the range of 0.05-1 µM in the reaction.
   	GC content in the primers in no appropriate	•      Check GC content of the primers •      Run gradient temperature PCR
   	Contaminated reaction mixture components	•      Use freshly prepared new reaction mix. •     Wear gloves and use sterile and clean filter pipette tips during setting up of the reaction.
   	Too low or incorrect annealing temperature	•      Recalculate primer Tm values. •     Increase annealing temperature, but intermittently.
   	Premature replication	•      Prefer using a hot start DNA polymerase
   	Template degraded, primers degraded or both template and primers are contaminated	•      Change the primers. •      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.

Additional Information