2 replies to this topic

[dnarna909]

I am using ligation mix to do the transformation, this vector is kanamycin resistence. After pick up the white clone into the LB medium, I am using alkaline lysis method to do the mini-prep.

I can't get any yeild from this bacterial. I can gurantee there is no problem with the solution, because control works fine.

There is some strange phenomenon in the whole process:

1. ligation mix transformation Bacterial is very white when it is growing in the tube, which is not like my control.
2.After add Sol ll , ligation mix transformation Bacterial stilll very cloudy, seems that no Bacterial had been lysis. I am sure there is no problem with Sol II, the control bacterial work well.

Is there anybody had this kind of problem? What should I do with this? This ligation confusing me too long a time.

[Vini]

dnarna909, on May 11 2009, 05:34 PM, said:

I am using ligation mix to do the transformation, this vector is kanamycin resistence. After pick up the white clone into the LB medium, I am using alkaline lysis method to do the mini-prep.

I can't get any yeild from this bacterial. I can gurantee there is no problem with the solution, because control works fine.

There is some strange phenomenon in the whole process:

1. ligation mix transformation Bacterial is very white when it is growing in the tube, which is not like my control.
2.After add Sol ll , ligation mix transformation Bacterial stilll very cloudy, seems that no Bacterial had been lysis. I am sure there is no problem with Sol II, the control bacterial work well.

Is there anybody had this kind of problem? What should I do with this? This ligation confusing me too long a time.

are you working on E.coli?? If yes, the colonies should not look white when growing in tubes, only that the LB medium will look turbid. One possibility is that u r working wid some sort of contamination. Please rule that out first. chk whether ur antibiotic (which u add in lb med.) is working fine. I might sound foolish   but I hope, u r pelleting down the cells and resuspending it in Sol. I n then only adding Sol II!!!

[hanming86]

Smell the tube.

if ur negative control smells different from ur sample to be tested.. then yea..

E.coli got a distinct smell. If u have never smelled e.coli, u aint going far. it's prolly the fastest way to "partially" rule out contamination.