I performed restriction double digestion of my plasmid with ECORV-HF and PacI with the fallowing reaction protocol : (Source : NEB Cloner)
Steps
COMPONENT :
Total 50 µl REACTION
1) DNA = 1 µg
2) 10X rCutSmart Buffer = 5 µl (1X)
3) EcoRV-HF = 1.0 µl (20 units)†
4) PacI = 1.0 µl (10 units)†
5) Nuclease-free Water = Make upto 50 µl
After incubating at 37 0C for overnight (12-14 hr’s) and running on 1% Agarose gel. I got 2 bands at their corresponding size, But one of the band is very faint, I’ld like to know what should I do so that I can see a clear band.
Should I :
1. Increase the incubation time to 24 hr’s.
2. Decrease the DNA amount.
3. Increase the Enzyme Amount.
I'm attaching the Pic of Agarose gel.
I want to give few suggestions
1. Re run the gel with a different agarose and increase the gel percent to 1.5 and see if there will be changes
2. If you stained before, attempt not to stain this time around and let the gel run before staining. If you stain before consider using another stain and loading dye
3. In some cases this faint band may be due to poor DNA quality. Consider quantifying again and repeat experiment
Agreeing with Bamidele Tolulope Odumosu, I guess prior to reattempting the experiment, you should re-run the gel with increased percentage. The yield of your digested product is too low; Please check if both the double digest sites are too close to each other. If that is the case, you may attempt a sequential digest, rather than a simultaneous double digest.
It is hard to say if you have a poor yield without knowing the relative sizes of the plasmid and the insert. For instance if your plasmid is 30kb and your insert 300bp then of your 1ug DNA total there is only about 1/100th of the total signal is insert. I would cut more plasmid and do not run the gel for very long ( 1/4 the time of your picture) then you will still get good separation of plasmid and insert but the insert will be more compact and clearer but do check what yield you can expect given the relative sizes and you may find that this is a very good result. remember also your total volume is 50ul and if you are only running a fraction of this on your gel then the total yield will be greater. The staining of dna is proportional to its size so the plasmid will always look stronger than the insert signal
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