4 replies to this topic

[laraka]

Hi,
I am trying to transform DH5alpha cells with an EGFP fusion construct. Even though I use the same conditions for both, I get colonies from the control plasmid but  plasmid with my insert always ended up with no colonies. I have checked the fusion construct on a gel, it looks fine. So what might I  be doing wrong? I use 5-10 ng plasmid DNA and do heat shock transformation, and incubate in LB media w/o Amp for 1 hr at 37 C, then plate them on LB-Amp media.

Any suggestions would be helpful.

Thanks..

LaRaKa

[HomeBrew]

If the control plasmid produces colonies, but the experimental plasmid does not, and the only difference between the plasmids is the presence of an insert in the experimental plasmid, it is possible that the insert encodes a gene that is toxic to E. coli.

It could also be that your experimental plasmid prep is not what you think it is, or perhaps the experimental plasmid is huge while the control plasmid is not.

[laraka]

HomeBrew, on 16 November 2010 - 01:00 PM, said:

If the control plasmid produces colonies, but the experimental plasmid does not, and the only difference between the plasmids is the presence of an insert in the experimental plasmid, it is possible that the insert encodes a gene that is toxic to E. coli.

It could also be that your experimental plasmid prep is not what you think it is, or perhaps the experimental plasmid is huge while the control plasmid is not.

I considered these possibilities. Size shouldn't be a problem in this case since the insert is 620 bp. Toxicity might be the reason, however the same construct was used last year in another study in the lab and now we wanted to replenish the stock but had this problem.

[perneseblue]

Is there anything recorded in the construction history on how this particular plasmids was initially grown in E coli?

[laraka]

perneseblue, on 16 November 2010 - 09:36 PM, said:

Is there anything recorded in the construction history on how this particular plasmids was initially grown in E coli?

Unfortunately there wasn't a vector map since the construct was made by a previous member of the lab. But I've checked the papers published by my lab and found out that the vector is a different one than we thought. Thanks  for pointing me to look at the records of plasmid.Finally I've changed the antibiotic and got colonies today