HI..
POSSIBLE REASONS MAY BE FOLLOWING
1. Gel shift.
Enzyme that remains bound to the substrate DNA will affect the electrophoretic mobility of the digestion products. This will result in a band or smear above the expected band. Use 6X DNA Loading Dye & SDS Solution for sample preparation or heat the digested DNA in the presence of 1X SDS prior to electrophoresis.
2. Contaminated reagents.
Digestion reaction components may become contaminated with nucleases due to improper handling or storage. Nuclease contamination causes DNA degradation, which appears as diffused DNA bands on a gel.
Perform four control reactions:
I – without restriction enzyme,
II – with a new vial of buffer,
III – without restriction enzyme, with a new vial of buffer,
IV – with commercially available water e.g. Water, nuclease-free. OR Freshly autoclaved double distilled water.
Hope this will help
Hi
Do the following and attach picture. though at 500bp your digested product is not visible.
lane 1: Marker. Lane 2 plasmid without digestion, lane 3 plasmid digested with xba only, lane 4 plasmid with bamhi, and lane 6 with double digestion, lane 7 if u have a positive clone having same restriction sites (ask any one in your lab).
Use restriction buffer as per recomendation of the company you are using.
i have assumed you have confirmed your clone by pcr..
if possible confirm more colonies.
Hope so this will help you.
Perhaps a silly comment but you have checked that Xba and BamHI don't cut your insert?
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