What should the starting template DNA quality and quantity be for PCR?
Both the quality and quantity of nucleic acid starting template affect PCR, in particular the sensitivity and efficiency of amplification. PCR sensitivity and efficiency can be reduced by the presence of impurities in nucleic acid preparations or in biological samples. These PCR inhibitors are completely removed when template is prepared using QIAGEN Kits for nucleic acid purification. Please refer to the Brochure "Maximizing PCR and RT-PCR success" for additional information.
The optimal primer–template ratio has to be determined empirically. If too little template is used, primers may not be able to find their complementary sequences. Too much template may lead to an increase in mispriming events. Generally, no more than 1 ug of template DNA should be used per PCR reaction. As an initial guide, spectrophotometric and molar conversion values for different nucleic acid templates are listed below.
Spectrophotometric conversions for nucleic acid templates
|1 A260 unit*||Concentration (ug/ml)|
*Absorbance at 260 nm = 1
Molar conversions for nucleic acid templates
|1 kb DNA||1000 bp||1.52||9.1 x 1011|
|pUC 19 DNA||2686 bp||0.57||3.4 x 1011|
|pTZ18R DNA||2870 bp||0.54||3.2 x 1011|
|pBluescript II DNA||2961 bp||0.52||3.1 x 1011|
|Lambda DNA||48,502 bp||0.03||1.8 x 1010|
|Average mRNA||1930 nt||1.67||1.0 x 1012|
|Escherichia coli||4.7 x 106*||3.0 x 10-4||1.8 x 108**|
|Drosophila melanogaster||1.4 x 108*||1.1 x 10-5||6.6 x 105**|
|Mus musculus (mouse)||2.7 x 109*||5.7 x 10-7||3.4 x 105**|
|Homo sapiens (human)||3.3 x 109*||4.7 x 10-7||2.8 x 105**|
* Base pairs per haploid genome
** For single-copy genes