After I run my pcr on agarose the DNA is still in the well
Posted 06 April 2012 - 11:43 AM
10x buffer 5uL
Plasmid DNA 2uL
Primer #1 2.5uL
Primer #2 2.5uL
I then run it for 30 cycles
Initial 94C 5 mins
Denature 94C 30 sec
Anneal 55C 1 min
Extend 68C 5 min it's 1.9 kbp long and pfu needs 1-2 mins per kb so 4 mins plus an extra minute just in case. Please note the pfu I am using works at 68C not 72C, this was a lab made pfu
Extra extend 68C for 10 mins
My agarose gel is at 1.2%
Can someone tell me what the fudge I'm doing wrong?
Posted 06 April 2012 - 01:46 PM
Have you tried serially diluting your PCR product bec if it's too concentrated, it can get stuck in the wells? Or you could have used a lot of template DNA and then you have carry-overs.
- hobglobin, personal comment about my beauteous photo......
Posted 07 April 2012 - 12:45 AM
And as casandra allready stated: did the ladder run?
This might sound stupid, but are you sure the gel (electricity) was on? Maybe it wasnt connected well....
ALways check to see for "air bubbles" to make sure its running the way it should.
Posted 08 April 2012 - 10:58 AM