Hello dear community
I was running my bacmid DNA samples and I ran for 01 hour in agarose gel. But after running for one hour I could not see a single band in my gel including my control which was bacmid suppported DH10BACY competent cell. I am wondering what could go wrong with this? Can you guide me in this situation?
Hello Sadia,
Since the ladder is visible, the gel and buffer are fine. Decrease the run time to 30 minutes and keep the voltage between 80 and 100 V. Also, I recommend you check the DNA quality and increase the sample amount.
Hi Sadia,
As Bacmids are usually very large they can be hard to visualize. Here are some additional recommendations:
Agarose Gel concentration should be 0,5%
Use TAE buffer (not TBE), however even if TAE has some advantages for bigger molecules it is more prone to overheating - you should be aware of this
Use low field strenght (1-2 V/cm (Voltage to be applied (V) = distance between the electrodes (cm) x recommended V/cm)
I would think a runntime for 30 min might be a little to short, expecially with the low field strenght - however you could check in between (but be carefull as these low percentage gel are not very stable and break easily)
For me it also seem that the staining of your gel is not very strong because the visibilty of your marker is not very good. If you stain after electrophoresis stain it longer or put Ethidium bromide directly in your gel.
As Mariel Polanco Valdez recommended I would also increase the sample amount.
Good luck
Dörthe Andrea Kesper Thank you so much for your multiple suggestions.Yes I used 0.5% gel and the gel is so soft and fragile.