PCR band disappears in agarose gel! - Agarose gel electrophoresis (Aug/15/2006 )
Hello all, perhaps you can help me with this one.
I did PCR on genomic DNA with primers and reaction conditions used succesfully before. I loaded 15ul of a (25ul) PCR reaction to a 2% agarose gel in 1X TAE with EtBr. The buffer in the box is 1X TAE no EtBr in buffer. Ran gel at 100V.
I checked my gel to see if PCR was succesful about 10 minutes after loading. Bands were visible not in all samples (this was expected). It had not run long enought to be able to read the ladder. Placed the gel back in the box and ran an additional 25 minutes. Checked my PCR on the UV box again and my PCR bands had disappeared, the ladder still present.
Can anyone help me identify what could have happened? Thank you in advance.
-izzy
How small is the PCR fragment? How bright did the band look after 10min.
Ethidium bromide runs in the opposite direction of the DNA so a small fragment which might have been visible, might b barely visible after running a few extra minutes.
Ethidium bromide runs in the opposite direction of the DNA so a small fragment which might have been visible, might b barely visible after running a few extra minutes.
The PCR fragment was supposed to be 425bp. The brightness looked similar to my ladder (100bp ladder from NEB)
your ladder is present while ur PCR is lost. well, i am also lost.
U could load the remaining 10ul and observe. Let us know what happened.
How small is the PCR fragment? How bright did the band look after 10min.
Ethidium bromide runs in the opposite direction of the DNA so a small fragment which might have been visible, might b barely visible after running a few extra minutes.
The PCR fragment was supposed to be 425bp. The brightness looked similar to my ladder (100bp ladder from NEB)
Well, I still think it is because that EB ran away. Adding some extra EB in the TAE (maybe you use TBE) buffer at the anode before running the gel may help.