4 replies to this topic

[SS17]

hope some one can help me on this one...

I have been trying to make plasmid preps of pbabe-neo-htert but always end up getting very low yield of the plasmid(~100ug/ml). I use quigen midi kits and they seem to be working very well with other plasmids that ive used.I grow the bacteria in LB with 100ug/ml ampicillin normally but tried lower concentrations like 50ug/ml thinking that the conc might be too high.However it doesnt seem to be a problem of bacterial growth as i seem to get descent sized pellets.I have no idea what could be going wrong here!

[perneseblue]

SS17, on Apr 22 2009, 06:21 AM, said:

hope some one can help me on this one...

I have been trying to make plasmid preps of pbabe-neo-htert but always end up getting very low yield of the plasmid(~100ug/ml). I use quigen midi kits and they seem to be working very well with other plasmids that ive used.I grow the bacteria in LB with 100ug/ml ampicillin normally but tried lower concentrations like 50ug/ml thinking that the conc might be too high.However it doesnt seem to be a problem of bacterial growth as i seem to get descent sized pellets.I have no idea what could be going wrong here!

How big is your culture?

And do you know if your plasmid is a low copy plasmid?

Does the plasmid express anything?

Does the plasmid contain any odd structures (ie tandem repeats)

The amp is not the cause. More Amp is better.

There are a few trick you can do,
1 - grow a larger culture. Grow two 100ml cultures. One might even go for a large 500ml culture in a 2.5L flask.

2 - decrease the incubation temperature from 37 C to 30 C. If the plasmid is making the cell unhappy/unhealthy a lower incubation temperature would help

3 - Add chloramphenicol into the culture at 14hr (after starting the culture) and harvest the cells about an hour later. Cm will stop cell growth but the plasmid will continue to increase. So you will get a higher plasmid yield.

4 - change the backbone of the plasmid to something more stable.

[SS17]

perneseblue, on Apr 22 2009, 07:27 AM, said:

SS17, on Apr 22 2009, 06:21 AM, said:

hope some one can help me on this one...

I have been trying to make plasmid preps of pbabe-neo-htert but always end up getting very low yield of the plasmid(~100ug/ml). I use quigen midi kits and they seem to be working very well with other plasmids that ive used.I grow the bacteria in LB with 100ug/ml ampicillin normally but tried lower concentrations like 50ug/ml thinking that the conc might be too high.However it doesnt seem to be a problem of bacterial growth as i seem to get descent sized pellets.I have no idea what could be going wrong here!

How big is your culture?

And do you know if your plasmid is a low copy plasmid?

Does the plasmid express anything?

Does the plasmid contain any odd structures (ie tandem repeats)

The amp is not the cause. More Amp is better.

There are a few trick you can do,
1 - grow a larger culture. Grow two 100ml cultures. One might even go for a large 500ml culture in a 2.5L flask.

2 - decrease the incubation temperature from 37 C to 30 C. If the plasmid is making the cell unhappy/unhealthy a lower incubation temperature would help

3 - Add chloramphenicol into the culture at 14hr (after starting the culture) and harvest the cells about an hour later. Cm will stop cell growth but the plasmid will continue to increase. So you will get a higher plasmid yield.

4 - change the backbone of the plasmid to something more stable.

Thank you very much for the reply!.The culture is approx 25 ml (as recommended by the company protocol). So yes I could try using bigger cultures.The plasmid is a high copy number plasmid. The backbone of the plasmid may not be a problem as such i think as i have a control plasmid with just pbabe-neo and ive been getting fairly good yields with that.But maybe as you mentioned it could be the insert itself thats being the problem  cos at around 3kb it is a quite a big one......

[Functional Screens]

1.  Sometimes even Qiagen's midiprep kit gives low yield of plasmid DNA
2.  pBabe-neo-hTERT is a little bit hard to handle (different sizes of colonies on the LB-agar plate).

My suggestions:
6ml LB culture per miniprep column with either Qiagen or Favorgen miniprep kit (elute DNA in 100ul)
100 ml LB culture per maxiprep column (use 10ml+10ml+10ml of soln 1+2+3 per each 50ml)
Note: grow your culture @37C for 300rpm for at least 16hr.

[Bettyboo44]

Can I just ask for a little help on this plasmid. It is great to have some hints on amplifying it thanks to all above. I am trying to amplify out the hTERT fragment to make a lentivector but I cannot find an accurate/reliable sequence for hTERT. I have found associated nt lengths of 3999 nt, 3500 nt (in this plasmid) and the ref of 3399 nt (http://www.ncbi.nlm....DATA=CCDS3861.2).

Can someone tell me where I am going wrong all I want it an expressible active protein.

thanks