I am planning to run some of the PCR products on a gel to identify the products of interest, perform a gel extraction, and do sequencing. My products of interest will be approximately around 300-550b in size. In order to get a band with a good resolution I have decided to use TBE instead of TAE and 2% of agarose. Do you think that 2% of agarose is too much or I should go for a low concentration (i.e 1.5%)? I'd also like to know the optimum voltage and time to separate my products (300-550b) in either 1.5% or 2% agarose gel. Please assist with the numbers.
Hello Davis,
Thanks. Well I'd use EtBr for staining and assuming my PCR works well, I will only expect a single band. But you never know. The sizes may vary from 300-550 bases (different per products). To get that band in the gel what should be the right voltage and time for a 1.5% or 2% gel.
Thanks
Hi,
I use 1% agrose with a voltage of 83 v and time less than 30 minutes.
Hi, how many products do you want to seperate? Two (300 and 550 bp)? Cheers, Nadine
I use 2% of agarose gel with voltage 50v around 30-40 minutes for band around 100 to 800 bp. I use the same protocole with EtBR or DNA stain (euromedex).
Hello Nadine,
I will be amplifying five different exons (5 PCR tubes) and sizes vary from 300-50bps. I just need an optimum voltage and time to run all 5 of these products in a 1/5%-2% gel. Thanks
http://www.biotechniques.com/multimedia/archive/00037/BTN_A_04374ST04_O_37093a.pdf
This might be an interesting read. They suggest low molarity buffers with low conductivity so that you can run gels faster, generate low heat, and have sharper bands.
if you use a 2% gel, you can get away with 120V and 30 minutes in TBE
The separation you will achieve will be comparable to that shown in the following picture for the bands from 500 to 100bp (I actually ran this ladder today with the above mentioned conditions, and the results were comparable to the picture)
https://www.neb.com/products/n3200-2-log-dna-ladder-01-100-kb
They use a 1% gel in this picture because they need to separate also the higher masses. Also, if you do a post run staining (instead of mixing the EtBr with the gel), your bands will be sharper.
Does anybody knows if the Gel-red get degraded? we run at 90v- 250 mA for one our, a 1.5% agarose gel, and we are having problems in visualizng the bands as well we don't see a good migration of the bads in the ladder. ! Is it probably due to the Gel-red we are using instead of ethidium bromide??
Tamara we never had this problem although we use GEL-RED all the time but make sure to cover the eppendorf or the flask that you put your GEL-RED in with aluminium or in a dark place , it´s senstive to light !!!
Many thanks David and Mohamad for your answers!!! all of them helped me a lot!!!!
Hi there,
I am wondering what voltage I should use and how long should I run my agarose gel electrophoresis if I am using 1xTAE buffer to look for bands at around 300 to 350 bp using 1% agarose gel concentration?
I often run it for 30 to 40 minutes at around 100-80V but I keep getting curvy lines.
Many thanks for your help!
For 1.5% agarose gel, I run at 100 volt and 80 mA for 40 minutes, and the result of band migration is very good
Hi, can suggest to me if I want to run electrophoresis of 1% gel what is the best voltage and time as we can observe a better separation of the ladder? HELP ME.....
We also observed some aberrant DNA and RNA migration with GelRed and GelGreen using Precast protocol (Agarose and PAGE). We solve this by using the post-staining according to the manufactures’ protocol. https://biotium.com/wp-content/uploads/2015/02/PI-41004-41005.pdf
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