Purify the fragment with a suitable purification method. All PCR products must be purified. Do not give them to us straight out of the thermal cyclers. Excess primers, salts, or taq, interfere with the sequencing reaction. Note: If you have more than one band in your PCR reaction, you will need to do a gel extraction to isolate the band you want to sequence. Multiple bands will ruin your sequence. When doing gel extractions, take steps to minimize agarose contamination and exposure to UV light.
- QIAquick Gel Extraction Kit. (cat#28704)
- QIAquick PCR Purification Kit (cat#28104) This will NOT work for sequencing if you have multiple bands! There are also problems with getting rid of excess primers. Optimize your PCR reaction!
- Wizard® SV Gel and PCR Clean-Up System
- Zymoclean Gel DNA Recovery kit ; (Protocol shared by Gina DiGregorio from Phillip Kern’s laboratory)
Dilute your PCR primers to 1.6uM (1.6pmol/ul) and provide 4-5ul for each reaction.
NOTE: The fact that a primer works for PCR does not ensure that it will work for sequencing. PCR reactions are exponential and can use newly created product as template. Sequencing is linear and creates many fragments of different sizes. They are not the same thing.
Estimate the concentration by running known standards with your samples or measure the OD260. Use the table below and adjust your templates to the appropriate concentrations. Provide 4ul for each reaction.
|Size (bp)||Dilute to:|