gel electrophoresis - eco r1 digest with lambda

[kazz143]

just want to know why if there are six fragments present in an Eco R1 digest of lambda then why are there only five bands visible on a .8% agarose gel? so where have the 21.2 kbp gone? is it to big to migrate therefore has not show up on the gel? can someone explain to me please? thankyou

[Moonbear]

A few possibilities come immediately to mind, but no way for me to know if any of them are what happened in your case. You may have gotten an incomplete digestion, you might have run the gel too long or too short of a time so either ran a band off the end or didn't get full resolution of all the bands, you might have had insufficient ethidium bromide, or it wasn't uniformly mixed through your gel, so couldn't detect a band, or your band might have been too faint to see.

[Charion]

Larger fragments are usually easier to see (more EtBr incorporation). However, 21.2 kb is pushing a 0.8% agarose gel. As a rule of thumb, the maximum resolution to be expected for a 0.8% gel is around 0.5-5 kb. Did you run a marker with a relevant size?Also consider that the overall gel size is also an issue. In mini-gels it is kinda tricky to resolve that size.
If the agarose is of good quality you can try to go down to 0.6%.

[kazz143]

hey thanks for ur posts. i understand now the 21.2 kbp is not the missing one. its becasue in an ecor1 digest of lambda it produce 6 fragments two of which are alomost the same length i.e the 5.6 and 5.8 kbp therefore we can only see 5 bands not 6 becasue the have migrated at the same pace to almost the same place donw the gel resulting in oine large band hard to tell that dere is actually two bands. thanks heaps newase