Strange things in clone - (Sep/29/2005 )
I construct a clone which carries linker for downstreaming application.
When I try to insert a polyA fragment to this vector, I encountered a problem that I got about 10 colonies. But none of them can get plasmid DNA. It is very strange.
During the preparation of plasmid DNA using phenol protocol, there is a srange phenomenon that between the phenol layer and water layer there are many white things I don't know. It seems that it is protein. But when I do second extraction of the upper layer, the same white things exit. I think it is impossible that there are so many proteins in my samples. After ethanol precipitation, I run gel to see the bands, no plasmids bands appear and only the RNA bands can see.
I have try transformation and pick the colonies and extract plasmid DNA twice. But from all the colonies I can not get plasmid.
What a strange thing. Can all of the colonies result from integration into chromosome?
BTW, I dephosphorated my vector and gel purified the two fragments. Then ligate and transform to DH5a cells. The control has no colony almost. And the positive plate has not so many colonies but 10 or so.
Can someone give me some advice?
Thanks in advance.
When I try to insert a polyA fragment to this vector, I encountered a problem that I got about 10 colonies. But none of them can get plasmid DNA. It is very strange.
During the preparation of plasmid DNA using phenol protocol, there is a srange phenomenon that between the phenol layer and water layer there are many white things I don't know. It seems that it is protein. But when I do second extraction of the upper layer, the same white things exit. I think it is impossible that there are so many proteins in my samples. After ethanol precipitation, I run gel to see the bands, no plasmids bands appear and only the RNA bands can see.
I have try transformation and pick the colonies and extract plasmid DNA twice. But from all the colonies I can not get plasmid.
What a strange thing. Can all of the colonies result from integration into chromosome?
BTW, I dephosphorated my vector and gel purified the two fragments. Then ligate and transform to DH5a cells. The control has no colony almost. And the positive plate has not so many colonies but 10 or so.
Can someone give me some advice?
Thanks in advance.
I had this problem zwo weeks ago and that was contamination on the plates not real E.Coli bugs and the cause was a dead ampicillin stock I did a new solution and plate againthe clones and none were growing so Yes it can happen and that explain why you don't get DNA out of your minipreps !
Pesji
What protocol are you using for your plasmid prep? Are you using alkaline lysis followed by phenol-chloroform? Are you performing a test digest on the plasmid to see if it is cut? I think we need some more info to help you out here.
DH5-alpha should be rec- so I thought they cannot integrate plasmid into genome...
pesji is right you should check your antibiotic, if it was good then perhaps check the pH of the phenol you are using in extraction, maybe that is your plasmid dna at the interface because the pH is too low... use pH 7-8 for DNA extraction...
HTH
As additionnal control you can try to run a pCR on the colonies to be sure that they are really E.Coli recombinant and not contaminated bugs !
Pesji
pesji is right you should check your antibiotic, if it was good then perhaps check the pH of the phenol you are using in extraction, maybe that is your plasmid dna at the interface because the pH is too low... use pH 7-8 for DNA extraction...
HTH
I picked the single colonies into freshly prepared 3ml LB liquid with Amp. But I can get bacterial culture.
If the colony in the plate is not E.Coli and something else because of the uneffect amp, why can I get bacterial culture?
Thanks
If the colony in the plate is not E.Coli and something else because of the uneffect amp, why can I get bacterial culture?
Thanks
OK you're right it doesn't make sense if your colonies are growing in liquid culture but on the other hand if it's the same ampicillin that you are using
Just try to plate your colonies on new plates with freshly made ampicillin it's used 50micrograms/ml in plates and 100micrograms /ml in liquid culture
Ampicillin is usually soluble in DD Water, make a new 1000X stock of 100mg/ml in DD water
and let's see what you get !
pesji