No growth after picking positive clones - (Oct/14/2007 )

Hi all

I am trying to clone a 900bp fragment in E.coli. I carried out blue white screening for positive clones. when i picked the white colonies and cultured in LB amp media, there is no growth at all. There are approx 200 well spread colonies (total). But seems like either the colonies have dried up or i dont no what else is happening. I have changed the LB media but still there is no growth. The incubation conditions are 210rpm at 37degrees centigrade. And there is no contamination on the master plate. Has anyone else faced this problem. Can anyone plz suggest what i need to do?

thanks

Hi,

is it possible that your construct is toxic for bacteria?

Do you really have an ampicilin resistence in your vector?

Hmmm....
1) Where your amplicilin plates old? How much ampicilin do you use in your plates?
2) How fast does your e coli strain grow? Some strains actually take 2days before a culture becomes cloudy.
3) How much ampicilin do you add to the LB medium?

If your cloned fragment is toxic or slowing growth, you could just pick a colony and put it straight into lysis buffer to extract DNA from it.

You probably wont get much DNA but if you run the toothpick (with the bacteria on it) on an amplicilin plate before you put it into the lysis buffer and let it grow o/n, you will have more bacteria to work with. If it turns out you have your desired clone, but not much DNA, you can just transform it back into E. coli.

Hope this helps!

First off, make sure your empty vector grows in LB with Amp selection. Amp plates are good at 4 C for about a month. Are you doing TA cloning? How are you purifying your insert?

I've found that if you gel purify your insert after digestion and leave it under the UV lamp for too long it trashes your DNA and problems like no growth in LB media occur. If you must gel purify your DNA, do it as fast as possible.

Another suggestion would be to subculture the positve clones on a fresh LB+Amp plate to see if your selection is really there. If it is they should grow on the new plate and then pick those colonies to grow in liquid.

That's where I would start.

Hope I was able to help.