Hello
We are working on a PCR product with an approximate size between 5000 and 7000 bp, we want to use it as a positive internal control, since we have sequenced it and it corresponds to the fragment of interest. However, when we purify it with ExoSAP-IT and use it for PCR amplification, when electrophoresis is performed there is a nonspecific banding and the expected product is hardly observed. Can you help me know what is happening?
Did you heat inactivate the SAP reagent? Also, what size product were you hoping to see from your PCR? You have a strong band at about 300 bp.
If I understand the problem right you pcr amplify the 5000 base product,exosap the product and then re amplify the product without gel purification or any other size sensitive purification.
If this is the case then I suggest that your original pcr primers have a sequence that can produce a G quadruplex either between the 2 primers or as a hairpin structure on one or both primers individually. ExoSap only degrades single stranded dna ( eg primers) but if your primers have formed either a intermolecular or intramolecular G quadruplex then this is double stranded and exosap will trim off the remaining single strand but stop at the double stranded sequence. This may result in your cleaned up pcr product having (for instance) 10-15mer shortmers so when you try the reamplification you have short primers that can anneal in many places and the short pcr products will amplify better than the long product even though the amount of short primer is lower.. It is probable that this reasoning applies to any primer dimer that occurred in the first pcr reaction but this seems less likely as most primer dimer will be completely double stranded
Rubí Santiago I use this step of the purification before the PCR for sequencing and I never observe this problem. I recommended the answer of dr Paul Rutland.
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