pcr smear

[vijaymicro]

Hello All
I am working on HIV pol gene. I am trying to amplify the gene since last few months. it was not working. Not the hot start is working. but it works for some samples for some time. when I used the same sample that previously worked, it worked once and then dint. For the other samples (new). there is big smear right from the well. I have tried the different sample size for the primary and secondary. It gives some samples worked at some concentration and some sampeles at another concentration.

Now I got the band in one sample which was bright but there was little bit smearing and others showed smearing right from the well.

The samples are already extracted and I cant redo the extraction as they are extracted in african country and then sent. so difficult to do the re extaction

[cita]

Hai,has anybody here dealt with asymmetric PCR before? What is the primer ratios that is suitable for asymmetric PCR?

[Aga]

Originally Posted by cita

Hai,has anybody here dealt with asymmetric PCR before? What is the primer ratios that is suitable for asymmetric PCR?

Actually it depends on the primer pair. I know I haven't helped you much
We used assymetric PCR several times in our lab and in each case the ratio was different. What actually are you trying to achieve? You can give me some general info so I may try to help, but still I think you'll have to test it empirically.

[xinlong]

I am also facing this problem. I have tried to use the same DNA to do ITS, it worked well; but it always appear smear band on the AT rich region. I have tried so many times to exclude any possible mistakes, but failed. Who knows?

[lazyyan]

so... where is the solutions? just change the new DNA?

[charumathi]

Hi
I have a problem in amplifying my gene . I did SOE and annealed the fragments using PR polymerase. but the annealed fragment was very less concentration. I used this as template and amplified using PR polymerase but getting a smear from well. what is the reason for this . I used ll polymerases and getting the same result.

[branthwaite]

I consulted the sage:
Smears the reason: Hydrolysis of the DNA making short DNA oligomers that act as Primers. DNA hydrolytic enzymes may be introduced by rogue bacteria or fungii. Work as clean as possible.
Why does one PCR will work perfectly and another inexplicably fail: Again bacterial DNA. In our case the PCR is to a phage insert so any contamination with bacteria that carry phage (all do) will bias the PCR towards smears.
Answer: "We" have managed to restore weak bands by re-extracting the tissue sample, do remove as much of the residual buffer so as to bias the PCR towards amplifying the gene of interest
Good luck.

[samsauma]

I am having a similar problem except I am just getting these rectangular blotches (grainy around the edges), and in one instance, I got my results but there were still these fuzzy squares directly below. Ladder is typically fine but it is doing that "smile" thing (leading edge seems to be migrating a little fast).

[branthwaite]

i posted on smears a while back.
after careful watching of what may go wrong I saw that the primers were placed in/on ice. The Ice is filthy with those black bacteria that reside in the water taps. Ask an electron microscopist. furthermore those black bacteria have been doing what comes naturally all over the lab especially tap handles. "Ok so how do they get into the PCR?" I hear you ask. Well, if at any time you handle screw top primers, or in fact any eppendorf tube, with wet gloves you will immediately syphon glove tip residue into the top of the tube. Leaving your primers in wet ice will result in the ice water creeping into the screw thread on the primers tube. enough said?!

[gautama]

Hi, sorry to bother you all, but maybe you can lend me a hand for my problem

I am looking at the soil microbiota as a whole rather than isolating specific bacterial species. In my DGGE profile the intensities of each band is about the same at each lane
(seen at the same row), that's what make my head aches. I was hoping to see a different profile lane to lane, but it seems they were all look the same! But from one band to another in the same lane/column have different intensities. I used 338F-GC and 518R. Any ideas how can I avoid two bacterial species from stopping at the same point in my DGGE gel beside using different primers?
Thanks for your kind answer...