Double bands in PAGE DNA

[goodhands?]

I am trying to get kinetics of one nucleotide incorporation;I used a 32P-5end primer (29-mer), hybridized with a template as substrate; differentdNTP concentration, at fixed time and DNA polymerase concentration. The reactionwas stopped adding the same volume of the Loading buffer (90% Formamide, 50mM EDTA,0.01% Bromophenol Blue). Samples were heated at 95 degrees for 3 min and thenput them on ice. Samples were loaded to Polyacrylamide Gel (7.5%Acrylamide:Bisacrylamide (19:1), Urea 8M, 1X TBE, 0.05% APS and 0.03% TEMED),the gel was resolved at 2400 V until the Bromophenol bye reached the bottom ofthe gel (45cm). Gel was dried. To quantify the primers and the products, one phosphorscreen was exposed to the gel.

 

At the beginning there was no problem, I could see just oneband in the negative control (no dNTP), and two bands when dNTP was added (Thehigher dNTP concentration, the higher intensity of the second band). This happenedfor at least 3 different experiments. And then I started to observe smear bands,and worst of all, was when I observeddouble bands in all the wells. After 1 month, labeling again, preparing alwaysdaily fresh loading buffer, new Acrylamide solutions, new TBE, new TEMED andAPS I could see I one nice gel with just one band in the negative control andtwo bands when dNTP was added; PROBLEM SOLVED???, NO; because then I run anothergel with previous samples (those that gave me the last nice gel) and new ones;and I observed two bands in all the sample, negative control include. So now Ido not know what to do to get again nice gels. Someone can help me with thistrouble?

 

Edited July 5, 2011 by goodhands?

[Arete]

Negative control lights up - are you saying you have a contamination issue?

 

Diagnosing the source of contamination over an internet forum is like giving haircut over the phone... have you cleaned/autoclaved your pipettes? What's your tip regimen? Who else uses your lab/bench space/reagents? Do you have clean and dirty labs?

 

The only real way to deal with contamination is to eliminate potential sources one by one until you find it unfortunately. Good luck.

[goodhands?]

Negative control lights up - are you saying you have a contamination issue?

 

Diagnosing the source of contamination over an internet forum is like giving haircut over the phone... have you cleaned/autoclaved your pipettes? What's your tip regimen? Who else uses your lab/bench space/reagents? Do you have clean and dirty labs?

 

The only real way to deal with contamination is to eliminate potential sources one by one until you find it unfortunately. Good luck.

 

 

Hi,

 

Yes, when I saw the double band in Negative control thefirst thing that came to my mind was this is a contamination issue, maybe dNTPin some buffer, but after labeling again (three different times) I still seeingthe two bands, and not even when I load just the primer without neither buffersnor DNA polymerase. So I don´t think that is because contamination.

 

Well the thing is that previous negative control that gavedouble band in a previous gel, and then reloaded in the last “nice gel” gave asingle band. But when I tried to reproduce the exact conditions of this gel andload the same negative control I observe again two bands on it. Well know I amtrying with others lab´s reagents.

 

Thanks for your concern.

 

[Tr0x]

My guess is that your dNTPs are contaminated. You said that "The higher dNTP concentration, the higher intensity of the second band" which immediately suggests some type of contamination in them.

 

dNTPs get contaminated fairly easily, as they're usually used by many people and for a variety of DNA work. dNTPs are also pretty cheap, so it should be easy to get a new batch.

 

Also, be very careful when loading your gels. You said that bands are occasionally showing up in all your wells (I assume you mean ones without samples in them too). Since screens for DNA are generally really sensitive, they'll pick up even the smallest bit of spillage into other wells.

 

A less likely reason for this would be different conformations of your DNA. Twists, hairpins or loops will travel through the gel differently, but there would have to be spillage to explain the bands appearing in the negative control lane.

 

 

 

[goodhands?]

My guess is that your dNTPs are contaminated. You said that "The higher dNTP concentration, the higher intensity of the second band" which immediately suggests some type of contamination in them.

 

dNTPs get contaminated fairly easily, as they're usually used by many people and for a variety of DNA work. dNTPs are also pretty cheap, so it should be easy to get a new batch.

 

Also, be very careful when loading your gels. You said that bands are occasionally showing up in all your wells (I assume you mean ones without samples in them too). Since screens for DNA are generally really sensitive, they'll pick up even the smallest bit of spillage into other wells.

 

A less likely reason for this would be different conformations of your DNA. Twists, hairpins or loops will travel through the gel differently, but there would have to be spillage to explain the bands appearing in the negative control lane.

 

 

 

 

 

 

Hi Tr()x,

 

First, when I said “in all the wells”, I really meant in allthe well that were load, sorry for that.

 

Second, when I said “The higher dNTP concentration, thehigher intensity of the second band”, this is because I am running a kineticsassays, so the higher dNTP concentration the higher dNTP incorporation rate bythe DNA polymerase (Of course, there will be a concentration point were thepolymerase will be saturated and the rate will be the Vmax). Also I have mycontrol without dNTP and with DNA polymerase and also gives two bands.

 

Yes, when I see a smear band, or a fuzzy one, I alwayssuspect of secondary structures of my primer. But the way that I am dealingwith this is: 1.-heat the sample at 95 Celsius degrees for 3 min, put them immediatelyon ice and then load them, 2.- run in denaturized conditions, in this case Urea8M, 3.- run the gel at 6O Celsius degrees.