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 :
- Add 25 µl 2X Master PCR mix into a PCR
- Add 20 µl of nuclease free
- 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.
- 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)
- Vortex samples for few seconds or mix the contents by pipetting up and down with a
- If PCR machine does not have a heated lid, add two drops of mineral oil into each
- 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)
- 95 °C for 1 minute (Denaturing step)
- 60 °C for 1 minute (annealing step)
- 72 °C for 1 minute (extension step)
Final extension Step:
- 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. |
Helpful Hint: In order to ensure that PCR conditions or thermocycler have no issues, run any positive control – known template and matching primers that work under specified thermocycler conditions
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