Hello all,
Trying to understand why my ladder and sample are moving at different speeds. Samples are from plasmid isolation of a 9kb plasmid before (left) and after (right) a PvuI digestion which should have created two bands, one about 6.5kb and one about 2.5kb. Running in TAE buffer using 1.5% agarose gel with SYBRSafe at 90V for about 45min. Ladder is NEB 1kb ladder located in 2nd and 8th wells (3ul). Any tips to improve band resolution would also be appreciated.
2 possible causes are that there is too much salt in the samples affecting the current in the gel and leading to slow running of the sample and
more likely the interaction of the sybr dye is making a big difference to the sample running characteristics. Try not adding sybr to the agarose and running the sample in an unstained ge. Then soak the gel in a solution of sybr safe before viewing and I would expect a large difference in the running speed. note ....the gel wil run about 10% faster in unstained conditions
Plasmid DNA is circular. Marker DNA are linear. Both types of DNA migrate differently. Your PvuI digestion is partial. It goes with the dynamics of interaction between water and DNA. Usually, single stands DNA or RNA let say carry more water, so it slows the DNA/RNA compared to double strand DNA. Plasmids can have supercoiled formation or open circle formation (nick in on of the strand that uncoiled the DNA). This is why you have multiple bands with you DNA prep.
Paul Rutland Thank you for taking the time to answer my question :)
Richard Villemur so am I using the wrong ladder? Do I need to linearize my plasmid to get an accurate size reading? Do you have any tips to improve my digestion? Thank you for taking the time to answer my question, I appreciate it :)
There is no good marker for circular DNA. For your digestion, improve DNA extraction protocol. Be careful with residual salt, SDS. Precipitate your DNA with ethanol, and wash the DNA pellet with 70% ethanol. Maybe your enzyme is old. Use another batch, or use another enzyme.
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