Im trying to identify good protocols. I get very faint bands.
I anneal at a 10:1 ratio of staples:scaffold.
I run a 1% gel at 70 volts in 1x TBE buffer using a 10000x sybrsafe green stain. I use 6x loading dye. Typically after 4 hours there is little migration of bands but there is a clear boundary (a couple of mm) between the aggregate which doesnt leave the well and folded, or partially folded, structures.
I run in a bath of ice.
Does anyone have any advice?
Hi, Simon.
I do not understand some of the non-scientific words in your question. I can only imagine what might happen. My advice followed.
1. Make your own fresh running buffer.
2. Wash your DNA by repeating precipitation of DNA and re-dissolve for a longer time at RT. The aggregates suggest dirty DNA or contamination of protein (protein extraction was not enough due to less volume of extraction sol compared to your sample amount. You can repeat extraction again in that case. Large and complex DNA mol needs much longer time to dissolve in H2O especially in higher concentration (Your imagination is necessary what is going on within the tube).
3. Spin the complete DNA stock sol at 3,000rpm for 3 min to precipitate any possible particulates and microscopic strings within the sol.
4. Take the upper part. Make a serial dilutions of a portion of DNA sol, and concentration check on the nano-drop machine.
5. Clean the gel chamber and dry, too
6. Run the gel with several dilutions in different wells.See the bands whether you solve the troubles.
7. You are handling tiny DNA molecules. They need much more attention in the macroscopic world of lab condition.
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