No colonies for transformation - (Aug/13/2014 )

I have been transforming my BL21 cells with my recombinant plasmid, however I never get any colonies in blue-white screening. I use ampicillin for selection. Most of the time for blue-white screening, I would observe faint blue patches, not colonies, on my plates, even on the controls.

After restriction digestion, the DNA would be positive on agarose gel, but after ligation, I digest them again to verify on agarose gel but I wouldn't get any bands. Could it be the competent cells or the ligation problem? I have tried using 1:1, 1:3, 1:5 and 1:10 ratios. 

I'm confused. Are you transforming already prepared plasmid (miniprep from a cell having the correct plasmid), or are you trying to construct the plasmid by ligation, and then transforming into BL21?

If the second, you will continue to have trouble, since transformation efficiency of BL21 cells is dramatically worse than typical cloning strains such as DH5a or Dh10.

phage434 on Thu Aug 14 03:14:21 2014 said:

I'm confused. Are you transforming already prepared plasmid (miniprep from a cell having the correct plasmid), or are you trying to construct the plasmid by ligation, and then transforming into BL21?

 

If the second, you will continue to have trouble, since transformation efficiency of BL21 cells is dramatically worse than typical cloning strains such as DH5a or Dh10.

I constructed the plasmid by ligation and then tried to transform into BL21. I used BL21 because I needed to express the fusion protein. Would using DH5a/DH10 affect optimal protein expression?

So, BL21 is an expression strain, and transforms quite poorly. When ligating DNA fragments, you should always transform into a cloning strain. When you have the correct plasmid in a cloning strain, miniprep the plasmid and transform prepared DNA into BL21 strains. Since you have millions of times as many correct plasmid molecules, the low efficiency of transformation will be of little consequence. Transform into DH5a or DH10B (or Top10, or NEB10, or any number of similar strains). It is not surprising that you see no colonies from direct transformation into BL21. Of course, there could also be many other issues with your ligation reaction.

Thanks! I will look into using DH5a. 

About ligation, I usually use the following:

T4 DNA ligase- 2ul

10x buffer w/ ATP- 2ul

Sterile water- 9ul

and, depending on ratios:

DNA Insert- 3ul

Vector- 1ul

We would incubate it at 16 degrees C overnight, usually about 17 hours. Is there any way to optimize ligation?

For ligations you want the backbone to have 20 ng or less in the whole reaction, which would usually be more or less undetectable on a gel, even if ran the whole ligation.

For the ratios, these need to be MOLAR ratios, not volume, ng amount or anything else. The formula for calculating a molar ratio is:

ng insert = ratio x (insert bp/vector bp) x ng vector

where ratio is a whole number, for instance a ratio of 1:3 would be "3" in the above equation. insert bp and vector bp are length of insert and vector in base pairs.

alright, I will try that too. Thank you!

hi i am a new member how to i send messages to people? it says i cannot do this :/

You need a minimum number of posts before you can do certain things, this is a spam prevention measure.