I am doing cloning of a big bacterial insert (3705bp) into a vectors of varying sizes ranging from 3017bp to 3469bp for my bacterial two hybrid experiment. Among other problems with my cloning I have noticed that neither 1% nor 1.2% agarose gel effectively separates my 1kb gene ruler ladder. So, I went for 0.7% agarose gel but I am facing an issue with the time required to run this gel. It runs so slower for me. It took me about 2 hrs (10Volts) to run it so that it crosses the center and reaches close to the bottom part of the gel.
I wonder that less agarose should make the gel run faster but why is it the opposite for me. Can anyone advice or give suggestions on potential factors to investigate or methods to improve migration speed or it is normal to take longer time.
If anyone has expertise in cloning big inserts in the bacterial two hybrid plasmids I would greatly appreciate some tips and suggestions to be successful in cloning.
Thank you in advance for your time and help.
Lower voltages give cleaner bands but take longer to run. With 90V it usually takes about 30 mins to run a 1% agarose gel. Also check what your amps are showing.
Dear Samiya,
1.0 or 0.8% (w/v) agarose gels should be perfect for your needs but the fact that you were running your gels at 10 V is a bit surprising. As suggested above, you should try running your gels at 80 – 120 V - the higer, the faster the gel runs, but also the hotter the gel gets and this can led to problems. You also need to make sure that the gels are made up in TBE buffer, and that the electrophoresis tank is filled up with fresh TBE too (TAE is an alternative buffer used in some applications).
Andrew
Thank you so much Christopher Bystroff , Andrew J Spiers and Robert Adolf Brinzer for your valuable suggestions to my question. The recommendation to increase the voltage to 100V -120V is duly noted although I wanted to provide a quick clarification regarding the voltage setting in my previous question.
Upon further inquiry in my lab, I discovered that the actual voltage used was indeed 100V, not 10V as indicated on the machine.
I appreciate your suggestion to increase the voltage, and it aligns with the correct operating conditions.
Moreover, I have stopped using 0.7% gel instead i am back to 1% and leave it to run for 45-60 mins to ensure a better separation in the above bands which seems to work well.
Although, I was not successful with the cloning and got empty plasmids :/.
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Molecular Methods
- Cloning