My clones disappear after subculturing! - (Oct/02/2008 )
I'm so happy i managed to find a forum specifically for cloning discussions. Hope someone will be able to make sense of this.
I've got a pcr product produced from a LightCycler run, about 500bp. The volume of the pcr product after its been through the LightCycler is 20uL, which I've purified.
I used the Topo TA Cloning system to clone my purified products into E.coli, which is plated onto LB+Amp plates and they grew nicely. I then pick one colony, extract its DNA (simple extraction - one colony into 10% chelex and incubate at 95c for 10min) and run it through the Lightcycler pcr to make sure the insert is there, and I got a good amplification curve.
So, very excitedly, I subbed whats left of the original colony into another LB+Amp plate to grow up pure culture...but nothing grew. Anybody who can shed some light on this?
hard to tell what ACTUALLY is wrong but here are a few suggestions
1. your amp plate could be too high of a conc?
2. your colony has no plasmid? ( unlikely since you could PCR out the whole thing)
3. what was being amplifed is something else?
4. the PCR product is toxic to the cell or has detrimental effect to the cell.
I bet you have more than one colony via TOPO cloning. I used to pick 3 -5 WHITE colonies and do plasmid prep and restriction digest to verify.
Could you by any chance subclone other than that particular colony ?
If you do it now prolly later today you will know .
Or culture em in LB broth with Amp ( 100ug/ml) that would be pretty quick 2 know. include a negative control to be sure.
Alternatively, might you have used plated your cells on an old LB plate, where the antibiotics have already degraded somewhat? Where there any satellite colony problems on the plate you picked the colonies from? Might the plate have been incubating a long time?
If yes, it is probable that the colony was a false positive. Did you PCR across the junction between the vector and insert? PCR is very sensitive and is able to amplify the left over DNA from the ligation mix present on the plate. If you amplified a segment within the insert, you risk getting false positives.
Do you have any other positive colonies that you can examine?
make sure the primers you use to pcr check for the insert is vector specific....topo ta cloning (M13 or T7 primers?) and not binding onto the e.coli genome dna showing false positive pcr results.