You should focus on optimizing these things before conducting a PCR:

1. Is your DNA template pure enough? Contain potential PCR inhibitors?

2. Is the size of your intended target large? If large, you need to use 'Long-ranged' DNA polymerase

3. Is your target GC-rich? If so, consider to use GC-rich PCR buffer, or consider to add PCR additive such as DMSO

4. Are your primers specific/unique to your target only?

5. Will your designed primers generate 'primer dimers' and influence the efficiency of your PCR?

6. What is the optimum Ta (annealing temperature) and extention time for your PCR?

7. Do you need to generate an overhang 'A' for TA cloning?

8. Do you need to use a High-Fidelity DNA polymerase? (If you want to clone a gene for expression later)

For optimization, there are

1) DNA extraction,

2) Primer design,

3) PCR setup as regarding component concentrations,

4) PCR cycling: denaturation, annealing and elongation temperature and duration,

5) choice of polymerase.

Please see the attached QIAGEN manual as an example. ;)

Hi, this might be also helpful: http://cshprotocols.cshlp.org/content/2009/4/pdb.ip66.full

Cheers, Nadine

You should focus on optimizing these things before conducting a PCR:

1. Is your DNA template pure enough? Contain potential PCR inhibitors?

2. Is the size of your intended target large? If large, you need to use 'Long-ranged' DNA polymerase

3. Is your target GC-rich? If so, consider to use GC-rich PCR buffer, or consider to add PCR additive such as DMSO

4. Are your primers specific/unique to your target only?

5. Will your designed primers generate 'primer dimers' and influence the efficiency of your PCR?

6. What is the optimum Ta (annealing temperature) and extention time for your PCR?

7. Do you need to generate an overhang 'A' for TA cloning?

8. Do you need to use a High-Fidelity DNA polymerase? (If you want to clone a gene for expression later)

Some nice answers already.  I would just add that preparing for PCR and real-time PCR are quite different- conventional PCR is a lot less sensitive to inhibitors and can handle primer dimers whereas real-time PCR is very sensitive and needs very good primer design.

All what you need, are already answered. I will add one more suggestion, be careful while desiging primer. you can not use the same primer for both RT and conventional PCR. The reason is product size and sequence type.

In case of conventional PCR you can design primer for the DNA sequences (intron, exons, UTR etc), but for real time PCR you need to design for the CDS sequences not for whole genomic sequences.

Thank you everyone. I got sufficient for mine from all of you.

You can please download pdfs of the files attached herewith to get certain interesting informations.