Good DNA quantity with nanodrop but nothing on the gel - (Nov/01/2008 )
Hi everyone,
I have extracted a plasmid on lots of growing colonies after cloning. I checked the concentration on the nanodrop andI get between 20 and 50ng/microliter.
I run them on a gel to see the size of the plasmid and to compare them with the control plasmid (with no insert).
But there is nothing on the gel. I have tried to load up to 10microliter, which would correspond to between 200 and 500ng loaded in the wells. But I still can not see anyband...
Can someone help me?
Thank you very much
xavier
I have extracted a plasmid on lots of growing colonies after cloning. I checked the concentration on the nanodrop andI get between 20 and 50ng/microliter.
I run them on a gel to see the size of the plasmid and to compare them with the control plasmid (with no insert).
But there is nothing on the gel. I have tried to load up to 10microliter, which would correspond to between 200 and 500ng loaded in the wells. But I still can not see anyband...
Can someone help me?
Thank you very much
xavier
Hi.
I can think of three things which could be the problem.
1) the nanodrop is a great instrument, but can easily give false readings. The cable could be damaged, there could be protein residue on the lens, etc etc. Try measuring a known (reliable) standard in various dilutions & see if the nano performes as it should.
2) can you provide more details about the gel. it could be going wrong there for a whole bunch of reasons (most likely, Ethid Bromide migrating in the opposite direction of DNA & your DNAS ending up in a part of the gel without EB).
3) I dont find 20-50 ng/ul alot. Are you sure you are actually successfully isolating plasmid DNA? Once aghain, more info would help.
How are you isolating DNA, what bacteria & what strain are you using, etc etc
You should be able to get at least 1 ug/ul if you use standard Coli's (eg XL1-blue / DH5alpha etc) and standard kits.
So, give more details & there is a better chance we'll be able to find out whats up.
20-50 ng/ul can be a lot if you have only a low copy plasmid. I normally get 60ng/ul out of a 100 ml culture with a low copy plasmid but 600ng/ul with a high copy plasmid. So it depends more on the plasmid than on the kits or cells he uses.
A gel should be able to show concentrations of 5ng/ul if stained with EtBr. Do you put EtBR to your gel or do you put the gel into a EtBr bath.
I agree that the Nanodrop could just show contaminations. What is the 260/280 ration and what is the AU260?
It makes no difference what the nanodrop says if nothing shows up on your gel -- another reason why I never bother with such devices.
hi,
thank you very much for your answers.
So, I work with E.Coli TOP10 and I use QiaGen Spin Miniprep or 5Prime kit.
And for the gel, I use SYBR safe, not ethydium bromide. So when I make the gel, SYBR safe is included.
When I do the miniprep on control coloniesm just the vector with no insert, I get a nice band on the gel. But when I do it with most of the recombinant colonies (which grow on Amp) so which do have the plasmid (with or without the insert), I have most of the times no bands... weird no?
thank you
We use SBYR safe too but sometimes I still stain it with EtBr in addition because SYBR safe is not as sensitive as EtBR.
Also I would recommend you to include a control in which you plate untransformed bacteria (so competent bacteria without vector) on the Amp plates. If they grow then you know that your Amp is bad. Did you buy the competent bacteria or did you make them yourself? If you make them yourself maybe they are contaminated with another Amp resistant strain.
Maybe your Amp concentration is not high enough.
The problem is that I have done miniprep on a lot of colonies, and with many of them, the concentration i get is around 20ng/ul and in a few, it is between 200 and 400.
When I concentrate DNA, around 90ng/ul and then run on a gel, I have a smear band from 10.000bp to around 500bp, but no clear band.
The nanodrop is working well, I have checked it with other samples.
It seems that I have a lot of background, bacteria with no plasmid.
I bought the competent cells, E.coli TOP 10.
I will try to transform again.
I use a shuttle vector to propagate the plasmid in E Coli before transforming B.megaterium with it. It has a tetracycline resistance gene. I will try to select with this antibiotic.
And I will also digest the ligation product with a enzyme that has a restriction site in the part of MCS that I have cut to insert my gene. Then, all the ligation products which will not have taken up my insert will be cut.
The smear could be genomic DNA. Maybe your kit is bad (maybe old). Did you try another kit maybe from a different lab?
dNTPs will give a signal on the nano-drop, if you have degraded DNA it will give a signal. I would guess that you have degraded DNA.