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Spot Test

 

A quick way to determine the concentration of for example fragments for ligation.

  1. dilute the DNA solution in H2O accordingly, best measurements are achieved in the 5-10 ng range
  2. mix in an Eppendorf tube:

    <table border="0" cellpadding="0" cellspacing="0" width="230"> <tbody><tr bgcolor="#ff9900"> <td colspan="2" align="center" height="20">Spot Test Mix</td> </tr> <tr> <td colspan="2" height="1"></td> </tr> <tr bgcolor="#ffcc66"> <td align="right" height="2" width="151"> </td> <td align="right" width="73"> </td> </tr> <tr bgcolor="#ffffff"> <td align="right" width="151">EthBr (1 mg/ml)</td> <td align="right" width="73">5.0 ul</td> </tr> <tr bgcolor="#ffcc66"> <td align="right" width="151">dil. DNA solution</td> <td align="right" width="73">x.0 ul</td> </tr> <tr bgcolor="#ffffff"> <td align="right" width="151">"H2O"</td> <td align="right" width="73">y.0 ul</td> </tr> <tr bgcolor="#ffcc66"> <td align="right" width="151">total</td> <td align="right" width="73">10.0 ul</td> </tr> <tr> <td colspan="2" height="1"></td> </tr> </tbody></table>

  3. prepare control samples (5 ng, 10 ng, 15 ng, 20 ng) using e.g. DNA from plasmid preps with known DNA concentration (prepare stocks, keep frozen at -20°C)
  4. spot the different mixes onto a saran wrap overlaying a regular UV transilluminator
  5. estimate the appropriate DNA concentration by comparing the fluorescence intensity of the test samples and the control samples

 

 

Remarks:

 

This technique provides a rapid way to make a rough but useful estimate of the DNA concentration of a given sample (e.g. isolated fragment). It is not highly accurate. Take care to make the comparison in the 5-15 ng range. Above 15-20 ng saturation is reached and no decent estimates can be made based on the fluorescence intensity. Furthermore, less EthBr intercalates in small DNA fragments (ca 0.5 kb) than in larger DNA fragments (3 kb). Thus, the method leads to underestimates of the concentration of small DNA fragments. Don't wait too long when making the comparison to avoid false results due to drying of the samples. Nevertheless, we regularly use this method for purposes such as cloning or probe labeling.