If I want to have a good yield of PCR without any unspecific bands and have my desired bands be strong, what should be the typical starting concentration of both my genomic DNA and primers in the PCR reaction tube? In another way, how many nanograms or microliters of the genomic DNA should I add to the 25 microliter PCR tube and what is the typical concentration of the primers to be diluted and how many microliter of the primers should I add?
First of all it depends on the specificities of your primers. Then, you can try to increase the annealing temperature re in order to reduce unexpected amplification products. Do you have primer dimers as well? I could say, start with 100 ng DNA per 25 microl, but in fact you should have an idea of the number of copies you have in the amount of DNA you add inside
Other wise you can make an agarose gel, separate the PCR fragments, cut the piece of gel containing the band, and then use a classical Kit such as Qiagen PCR purification kit it in order to extract the DNA from the agarose block containing the band that you have cut. It will remove ethidium bromide as well.
Good luck..
JL
Optimal concentration also depends on buffer, dNTPs (each has the potential to chelate magnesium) and MgCl2.
HOWEVER, you should only check if the A260/280 ratio of your sample is about 1.8. Then, do the PCR with 100 ng of DNA with final concentration 0.1-0.5 µM of each primer.
A primer concentration range that is commonly used is between 200-400 nM final concentration (the volume is not really important, keep it around 1 ul per primer). Regarding the amount of DNA for your PCR, you should really test it. There is no perfect answer, it depends on your assay. You could start from 100 ng total and do 1/2 dilutions to verify if you have inhibition of your PCR reaction at certain DNA concentration.
Hope this helps.
CL
there is some necessary rules in order to get strong and sharp PCR product band without non-specific bands,
1. Primer design is the most important step, and if properly it has done you will have a easy way toward your purpose and getting sharp band. specificity of primers is very important and some condition include GC content, Tm, and homo-dimer and hetro-dimer formation must be considered. you can use Oligoanalyzer website for analyzing of your primers.
2. PCR program
3. PCR components volumes must be adjusted.
For my PCR I used 10 pM primers (0.5 ul of each) and 50ng/ul of DNA and depends of reaction i use 1-3 ul. I did reaction for 20 ul so 0.5 ul of polymerase 2ul of buffer and 1ul of dNTPs and rest was ddH2O.
30 ng/ul DNA and 10 pmol/ul from each Primers (0.5 ul) are enough.
You can use from this PCR reaction:
DW: 18.85 ul
PCR Buffer: 2.5 ul
MgCl2: 1ul
dNTPs: 0.5ul
F primer: 0.5 ul
R primer: 0.5ul
Taq: 0.15 ul
DNA: 1ul (30 ng/ul)
Total: 25ul
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