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Cant see ppt after ethanol precipitation?! - (Jul/07/2008 )

HI! Since I was not getting colonies after transformation with the ligation mix, I decided to ethanol precipitate the insert and the vector together and then add ligase(just so that the ligase can actually see the two together, and this has worked for me before)...but ...after adding insert and vector (total DNA=500ng), I did not see any pellet after etoh precipitaion! I added 1/10 vol of 3M NaAc, vortexed, added 2-2.5 vol ice-cold100%etoh,vortexed, kept at -70C for 1 hour and then centrifuged at 14000rpm for 5min...nothing! huh.gif
...OR IS IT JUST ME GOING CRAZY??! dry.gif

-biorules-

QUOTE (biorules @ Jul 7 2008, 10:15 AM)
HI! Since I was not getting colonies after transformation with the ligation mix, I decided to ethanol precipitate the insert and the vector together and then add ligase(just so that the ligase can actually see the two together, and this has worked for me before)...but ...after adding insert and vector (total DNA=500ng), I did not see any pellet after etoh precipitaion! I added 1/10 vol of 3M NaAc, vortexed, added 2-2.5 vol ice-cold100%etoh,vortexed, kept at -70C for 1 hour and then centrifuged at 14000rpm for 5min...nothing! huh.gif
...OR IS IT JUST ME GOING CRAZY??! dry.gif

You don't see much pellet with 499ng. You just need to have faith in the religion of molecular biology and process the empty tube!

Anyways, next time add glycogen/glycoblue if you want to be sure.

-cellcounter-

QUOTE (cellcounter @ Jul 7 2008, 11:18 AM)
QUOTE (biorules @ Jul 7 2008, 10:15 AM)
HI! Since I was not getting colonies after transformation with the ligation mix, I decided to ethanol precipitate the insert and the vector together and then add ligase(just so that the ligase can actually see the two together, and this has worked for me before)...but ...after adding insert and vector (total DNA=500ng), I did not see any pellet after etoh precipitaion! I added 1/10 vol of 3M NaAc, vortexed, added 2-2.5 vol ice-cold100%etoh,vortexed, kept at -70C for 1 hour and then centrifuged at 14000rpm for 5min...nothing! huh.gif
...OR IS IT JUST ME GOING CRAZY??! dry.gif

You don't see much pellet with 499ng. You just need to have faith in the religion of molecular biology and process the empty tube!

Anyways, next time add glycogen/glycoblue if you want to be sure.



Yeah, that's exactly what am doing now! i thought i did see something at the bottom(hopefully) and am going with that...hailing all the gods of molecular biology !! tongue.gif Thanks anyway, cellcounter! biggrin.gif

-biorules-

QUOTE (biorules @ Jul 7 2008, 09:15 PM)
...just so that the ligase can actually see the two together...


hum... that sounds a bit weird... you know, everything is moving at the molecular level, and the ligases don't have "eyes"...

why not starting with your cleaned insert on one hand, your vector in the other, and put the water and mix, and put the buffer, and put the ligase, and mix and incubate and and and... really, you are only adding another step that could go wrong when you precipitate your insert and vector together, and then you might not be sure you have the same vector: insert ratio as you started with, you might have lost some during precipitation...

keep it simple. and consistent, you don't optimize a ligation by precipitating insert and vector together if it doesn't work, you change the ratio, or do something more well-considered...

-ph3no-