Im using terrific broth(TB) to grow e.coli for recombinant protein expression.it take minimium of 3 hrs to reach OD=0.5 at 600nm. what might be the possible problem behind this?
Dear Pushpa
just some question:
did you start your culture from O/N pre-culture or from a single colony from agar plate?
In the first case how much you dilute you preculture?
Did you growth the bacteria in wtih flask? at wich temperature and shaking rate?
Are you using and antibiotic for plasmid selection? Which antibiotic and which concentration?
All this details can drastically change the time that you need to reach the OD.
best regards
manuele
Why do you think, that 3 hours are a problem? That seems like OK to me.
Of course, if you'd started from OD 0.5, you'd reach it in 0 minutes. Of course that's non-sense, but hopefully you'll get my point - the higher OD in the beginning, the faster you get to the end. However, the more bacteria you introduce in the beginning, the more variation you may potentially have.
At least one think you can control - what you inoculate with as mentioned by Manuele. If you use colony from a plate from fridge or ON culture in small volume, that has already saturated, it will take some time untill they "wake up" and start dividing. What you may do is either to grow the pre-culture in the morning for shorter time (but I suppose you do not want to do that) or grow it over-night in larger volume. I usually use 10 ml instead of 2 ml. In that case the bacteria should grow for longer and you may catch them when still actively dividing (unless you start your pre-culture at 2PM of the previous day;)
I think Manuele asked right query, because to obtained a fixed no of cells depends upon various conditions. So, if you have already an overnight grown primary culture, then just add approx 500 microl into 50ml LB media, incubate at 37 degree C for 4-6 h. I think you would be able to get your desired concentration of cells.
Are you talking about your primary culture or secondary culture? If it is for secondary culture in T.B., 2% of O/N grown inoculum of E.coli would reach an O.D (600) of 0.6-0.8 in 3 hours. (provided you ensure proper aeration and agitation to the culture).
I have started with my primary culture(luria burtani broth) then tranfer it to secondary culture (terrific broth). For primary culture ,I pick ed single colony from my agar plate and then put it in steriled tubes containing 5ml LB media with ampicillin 50mg per ml. then i kept it overnight at 37 degree for 18 hrs with 125 rpm.50mg per ml.
For secondary culture also I used ampicillin.TB broth is incubated with the primary culture.
after that the culture is incubated at 37 degree untill OD reach 0.5.final the culture is induced with 0.6mM IPTG and kept overnight at 20 degree shaker.
This is my protocol i have been doing. but the problem is there is no uniform time period for the particulate OD.
Before i used yeast extract from FISHER SCIENTIFIC. with this ,i reach the OD at 2hr. Now i have purchase new yeast extract with same company ,here I reach the OD at 3 and half hrs. And i was think why their is so much different in time?
incubation at 37 for 18 hr & 125 is also problem. it should for only for 12 hours. It might be creating variants, death of some bacteria.
You should get OD at all time in 2-3 hours always.
Yeast extract batches may vary. That was also the motivation behind auto-induction media.
http://www.ncbi.nlm.nih.gov/pubmed/24203322
I'm not sure, if this one is the one, I don't have access to this.
So this may be an option for you. Obviously the "problem" is in the yeast extract (if nothing else changed), thus you can either try different batch, or defined medium.
I think you should measure the OD of overnight grown primary culture (LB). Then from primary culture add that much amount into your secondary culture (TB) that OD of secondary culture at 0 time interval will be 0.05. Now keep your secondary culture for incubation at 37 with 200rpm. You will get 0.5 OD in 2 hrs. You can also check OD of culture after 1hr to predict the exact time. Hope this will help you.
I have one more question. while measuring OD using spectrophotometer, should the blank solution be distilled water or medium that I'm using .
I prefer medium, as that will give you exactly the OD of your culture. However, the difference from water is not so big. You can try it by yourself.
- Badges
- Science topic
- Similar topics
- Biological Science
- Bioscience
- Biotechnology