help - (Feb/22/2010 )

hello, I'm having some problems in getting colonies after transformation :rolleyes: . Never inactivate the T4 DNA ligase but always froze (-20 ° C) ligations before making the transformations. Is freezing sufficient to inactivate the T4 DNA ligase?
Thank you all.
Ps-I did ligations with molecular ratios 3:1 and 6:1 on a volume of 15uL and DNA concentration around 20ng/uL (in DH5alpha competent cells).
sofiap

-sofiap-

sofiap on Feb 23 2010, 03:17 AM said:

hello, I'm having some problems in getting colonies after transformation <_<

. Never inactivate the T4 DNA ligase but always froze (-20 ° C) ligations before making the transformations. Is freezing sufficient to inactivate the T4 DNA ligase?
Thank you all.
Ps-I did ligations with molecular ratios 3:1 and 6:1 on a volume of 15uL and DNA concentration around 20ng/uL (in DH5alpha competent cells).
sofiap

/

1.Have the buffer you use thawed and mixed completely?
2.Have you put the ligation in proper temperature and for sufficient time? such as 16 degree O/N.
3.Are your competent cells efficiency high enough? Transform a plasmid as a postive control.

Hope those useful to you, good luck

-Cruise-

Maybe freezing is your problem? I have never even inactivated the T4, just gone ahead and transformed. Sometimes, though, what you're trying to transform in can be lethal to the cell so you don't get any colonies. Are you allowing them to recover in antibiotic-free media before you plate them? Are your positive controls growing?

-microgirl-

I ligate at RT for 30min. and directly use it for transformation. The main thing, is to make sure the ligase buffer is not degraded.

-scolix-

Hi, thanks you all for the tips.
1st- Yes i'm sure that buffer are completely thawed and mixed, although not completely sure if it is working as it should be (maybe this is the problem)
2nd- I did ligations at 16ºC O/N, O/W, 2h RT and results were always the same
3rd- my competent cells are recent and I tested the efficiency (about 8.9x10^{6 colonies /ug of DNA of plasmid) but I didn't positive control of my transformations (I do next time)

4th- Before plate, I put them in LB medium antibiotic-free for an hour with gentle agitation.

Next time I will do positive control and I'll try to check if buffer is not degraded.

Thanks

sofiap}

-sofiap-

Bad ligase is almost always the problem with cloning in our lab. We had an undergrad spend 6 months trying to clone something only to discover that the ligase was bad. It is sometimes worth a call to the vendor to make sure your batch of ligase hasn't been "recalled."

-microgirl-