Growth of E.Coli culture - please help out-i am desperate (Nov/02/2008 )
QUOTE (swanny @ Nov 9 2008, 11:49 PM)
QUOTE (SYDNEY @ Nov 8 2008, 01:11 PM)
MC1061/P3 Ultracomp WAS TEH STRAIN OF E.Coli used for transformation.
Great. What can you tell us about the plasmid? How big is the backbone? For that matter, how did you get hold of it (was it left to you by a previous student?)? If it was given to your supervisor, she should have the details from whomever sent it to her.
Your gel looks like a fairly standard miniprep. The different lanes show plasmids with differing amounts of supercoiling and nicked DNA. What is the size of the top band of the marker (it's hard to know unless you know which brand of 1 kb ladder you use in your lab).
You might want to try 50 ug/ml ampicillin final concentration, rather than 100.
Hi,
The vector is pCDM8 and the 1 kb ladder is from promega.
It has been sucessfully transformed in the past but it sbeen long time so the culture i relesed teh plasmids was a bit old.
Does teh culture gets of with the passage of time? Remember that i have told you intially that after transformation the colonies were grown on the plates but when i picked
teh colonies and cultured, growth was there still but when i released teh plasmids and purified, the gel i run is there.
teh ampicillin concentration used was 100mg/ml and tetracyline 2mg/ml as i already meantioned.
I think i need to do transformation again with some good plasmids and see what happens next.
-SYDNEY-
What is the size of pCDM8 meant to be?
Your initial amp concentration is 100 mg/ml, but what is the final concentration? The starting conc is only of academic interest, it's the final conc that really matters to the cells, and therefore to the success or otherwise of your experiment. Same goes for the tetracycline.
Take some of the DNA you prepped and cut it with an enzyme that is known to only cut once in CMD8. When you run that on the gel, you'll have an accurate idea of the actual size of the plasmid you have prepared.
Um, I just checked Vectorpedia (http://www.addgene.org/pgvec1?f=v&cmd=showvecinfo&vectorid=3388) and it has no mention of any antibiotic resistance genes (unless I've gone completely ga-ga!). You are sure about that name?
-swanny-