- The experiment we conducted involved digesting bacterial plasmids from two cell cultures (Bacterial cultures of NFκB2-Ex3-sgRNA CRISPR/Cas9 and pSpCas9n(BB)-2A-GFP)
- Materials used: Plasmid miniprep kit component, FastDigest BbsI, FastDigest AgeI FastDigest Green Buffer (10X), Nuclease-free water and Plasmid DNA, 1 μg
- Incubation: 37 °C in a heat block for 30 min
- Gel electrophoresis: pre-cast 1% DNA agarose gels that contain FluoroSAFE dye, 120 Volts for 40 min.
- Loading amount: 15 μl of digested DNA fragments
- This gel was shared between two groups (we're university students). The first and fifth lanes were 1kB DNA ladder: NEB N3232S) and the streaking pattern was observed in both group's markers and lanes.
- We believe it may be an issue with the electrophoresis machine itself or the pre-cast gel but would like to get a second opinion. Our professor couldn't make heads or tails of it.
It looks like a combination of too much sample and running buffer too dilute ( or in extreme cases gel made with water not TBE. You can also get this effect using samples with too much salt in them which can be caused by loading dye made up in too strong buffer. If you are not adding loading buffer to your ladder before loading on the gel then the fact that your ladder also shows this effect suggests too much sample or gel made with water as stronger possibilities
Hi, too much sample loaded into the well, Please reduce the amount and voltage used..
Hi Christian,
I suggest you to take just half of the volume per well of gel. In addition always run your gel between 70-80V range. One more thing i wish to add is monitor the buffer temperature, above 40 degree C may hamper the integration of gel subsequently resolution may affect.
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