Why cannot I get the colony in LB plate? - (Jan/31/2007 )
Dear all,
I am making library of some groups of SQR genes. Actually I separated them into 3 groups. I am trying to select some colonies for sequencing. I did experiment with 3 groups in the same time, same conditions (including LB plates, ampicillin, temperature.....) With 2 groups I did normally. But with the other, I failed. I repeated experiment in 3 times and I got the same result. I donot know why.
Guys, anyone has experiment in this situation, plz, explain for me. Maybe my interesting gene codes for a toxic protein, so it will kill the component cell?????? 
Love,
Jikji.
I am making library of some groups of SQR genes. Actually I separated them into 3 groups. I am trying to select some colonies for sequencing. I did experiment with 3 groups in the same time, same conditions (including LB plates, ampicillin, temperature.....) With 2 groups I did normally. But with the other, I failed. I repeated experiment in 3 times and I got the same result. I donot know why.
Guys, anyone has experiment in this situation, plz, explain for me. Maybe my interesting gene codes for a toxic protein, so it will kill the component cell??????
Love,
Jikji.
If you're concerned about a toxic protein, you could clone into a plasmid with very tight control of expression, like the pET pLysE plasmids (but I'm sure the BioForummers can give an extensive list from experience). If it's really bad, you might have to go to using an intein-based expression system, and make the protein in two halves in two different cells.
Just think of the huge range of skills you'll develop...
SQR gene = sulfide-quinone reductase. That means it is a functional gene.
But I sequence only one fragment of this gene, so I donot know how it is toxic or not with E.coli.
I am so sorry that I am in the dark of the expression of protein. I just want to harvest the colony for sequence. I expect someone can give me some way (donot need to check my gene is toxic or not) to resolve this problem. Thanks much.