I extracted gDNA from blood samples preserved on FTA paper using QIAamp DNA mini Kit and then ran PCR. The Agarose Gel Electrophoresis reveal expected band. To clean my DNA before Sequencing, I then used Zymo DNA Clean and Concentrator. But to my utmost surprise, there was no more band in the gel of the Agarose Gel Electrophoresis conducted after the Cleaning. What could have gone wrong?
So the three most likely times that this can happen are dna binding to the column. DNA elution from the column and not adding ETBR to the gel so any dna in the agarose remains invivsible. Try another sample, Measure the OD260 in binding buffer and again after binding to the column. If the eluate is clear of dna then the dna has bound to the column. Then elute and measure the od.Check that the amount loaded is roughly the same as the amount eluted and if not elute again to get a better yield If at this step you get dna eluting then the column has worked so the problem is in the gel so be sure you have etbr in the gel and that you do not run the gel for very long to make sure it does not run off the end of the gel
Dear Olayinka
which was the size of your pcr?
i'm not an expert of the Zymo kit
but generally for other standard pcr purification kits as qiagen, thermo or promega the complete lost of the pcr sample may happen if:
- pcr is too big and kit is not efficient for this size:
- problem in some buffers (eg forgot to add ethanol or isopropanol in the washing buffer)
Gel extraction are generally little bit more tricky because you need to be sure that all the gel is dissolve and that the buffer used for the gel eg tbe do not affect to much the ph of the binding mix.
Ciao
Manuele
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