pET14b vector with insert transformed in BL21(DE3) RIPL cells - (Nov/05/2012 )

Hi again,

A question came to my mind and thought I should ask about it to know the answer ahead of time

After step 6. of my protocol, the culture I get (that is the one I incubated with vigorous shaking at 18C for 16-20hr) can it be stored for a couple of days? I want to use it to get total cell protein fraction, the soluble and insoluble cytoplasmic forms and run on SDS-gel, but I do not want to do it the next day after the 16-20hr incubation. Is there a way to store that culture for a couple of days and continue my work later with it?

Thanks everyone

I usually spin it down and discard the media and then freeze the pellets at -20 until I have time to analyze them.

Andreea

Perfect idea Andreea. Thanks

For analysis, I will be doing SDS-PAGE with coomassie blue staining first, then Western Blotting. I will focus on SDS-PAGE now. I noticed different protocols online. I do not know which is best. I think it also depends on the protein size. My protein sizes are 16.52KDA and 21.38KDA. Now, what do you normally prepare in terms of the sample loading buffer concentration, running buffer, stacking gel ,and running gel? Oh and do you normally prepare ahead of time and store at a certain temperature, and use them when needed.

Sorry all this is new to me, so please bear with me.

In your case, I would use the 15% SDS-PAGE gels as described in Sambrook and Maniatis molecular cloning: http://www.amazon.de/Molecular-Cloning-Laboratory-Manual-Vol/dp/0879695773

Loading buffer I prepare and aliquot in 1 mL Eppis at -20; the rest I keep at RT. I never prepare gels ahead of time and then store them. People do that and keep cast gels in the fridge but over time the H+ gradient between the stacking and the resolving (which are usually at different pHs) will diffuse and the resolution of the gels will be sub-optimal.

Andreea

Ok hmm I can't really access the manual..hehe..do I have to buy it inorder for me to see its contents? Is there another way possibly.

Hello again,

So I did my step 1 of my protocol yesterday (transformation and plating). The plates have different plating volumes (100uL, 200uL, 300uL, and 400uL). I am not sure if my transformation worked the way it should. I mean I have ALOT of small colonies on my plates. So do you think I should move on to step 2 today? And if so, do I choose any single colony from any plate and inoculate that? Please respond back to me on this.

Thanks again :)

When you're transforming bacteria from purified plasmid DNA, you should get tons of colonies (unless your DNA or cells are poor quality).

There shouldn't be too much colony-to-colony variability in expression, so you can pick any colony and it should be okay. I usually inoculate 2 colonies in 2 tubes whenever doing a new growth in case one of them doesn't grow (for whatever reason). I would avoid any colonies that look significantly different from the others (may be a sign of contamination).

Thanks John That actually made me feel better about my transformation. Okay I will do just like you then, I will inoculate two colonies. Thanks for the hints.

All the best to you

I am still confused about OD600 measurement, and I am worried that I did it wrong last night.

I will clearly indicate my steps. I am using a cuvette spec.

For blank, I took 1ml fresh medium and diluted by adding 3ml water
For bacterial culture samples, I took 1ml of bacterial culture and diluted by adding 3ml water as well.

The cuvettes I am using are ones that light beam can pass through all sides. I made sure it is clean.
I loaded 1ml of my diluted samples in the cuvettes.

I blanked the machine and Abs. was zero.
I measured my diluted samples which I got negative Abs. for them

Looking at the cuvettes it seemed like the 1ml was not enough and I thought to myself that light is possibly not going through the solution at all to give a reading.

I ended up doing it again by filling cuvettes with 3ml of the diluted blank and the diluted bacterial culture samples (prepared above).
For blank I got zero.
For samples since I had two differet samples, one I got Abs. 0.321 which I multiplied by 3 (dilution factor)= 0.963, and the second one I got an Abs. of 0.360 which I also multiplied by 3 (dilution factor)= 1.08

Based on these Abs. I moved on to the induction step.

But I was thinking about it the whole night and I have a feeling that I am not correctly measuring OD600.

Please help me out, I am stressed about this.

Thanks all

Sorry in addition to the question I posted earlier about OD600 readings, I also have another question about the future steps. I remember in one of the posts Andreea mentioned that after a successful induction I can spin down (centrifuge) my culture to obtain pellets that can be stored at -80C until analysis. For analysis I wanted to compare the total cell protein fraction in parallel with other fractions such as the medium fraction, soluble cytoplasmic fraction, and the insoluble cytoplasmic fraction for the recovery of my target gene. In the pET manual 11th edition, there are protocols on how to isolate those fractions and then analyze them by SDS-PAGE. I wanted to follow their protocols since they are well written and explained but the problem is I won't have time to do it on the same day. I would have to as Andreea mentioned spin down the cells and store the pellet at -80C. If that ends up to be my case, can I still use my stored pellet to follow their protocols in the manual. I am worried since in their manuals, the protocols for the extraction of the fractions mentioned above always have as their first step: Prior to harvest take a 1ml aliquot of the culture..etc..
And this is not my case since I harvested the cells (pellet at -80C). What do you think I would have to do?
I hope I was clear enough I am sorry if this is confusing.
Thanks