Ran two Long Amp PCRs, one using un-purified template and the other a purified template. Un-purifed produces a PCR band and the purified doesn't.
Negative control has no band or smears in it. Unsure what could be the cause.
Reaction set up is 1x25ul with un-purified using .25ul of template and purified using .5ul of template. Using NEB LongAmp Taq2x Master Mix and nuclease free treated water. Also wiping down my work area before setting up to avoid contamination.
Still getting smears for my purified product, have use the same template using In Vitro Transcription to make m-RNA and there is no issues there. Unsure what the cause is.
May be ethanol contamination, after washing with ethanol, you can try to dry the column at 50degreeC for 10 minutes before elution to ensure there's no ethanol residue
I'd guess that you're getting carryover reagents from your template cleanup that inhibits PCR. So instead of getting a lot of full-length amplicons, you get a mixture of variable length fragments after PCR and they show up as a smear on-gel.
Maybe incorporate an extra alcohol wash to your purification procedure, reduce the volume of template that you're adding to your PCR reaction, and/or increase the extension time so that your polymerase has a better chance of amplifying your entire target sequence.
Did you check purified product before running PCR reaction? It might be caused by any of the purification steps, e.g., washing or elution step.
May be ethanol contamination, after washing with ethanol, you can try to dry the column at 50degreeC for 10 minutes before elution to ensure there's no ethanol residue
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