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Problem with PCR


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#1 Bioteck

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Posted 17 May 2004 - 01:01 PM

Hi.-
I have a problem with amplification of DNA by PCR. The extraction of DNA is direct from plant. i use PVP for keep out of DNA Polyphenols from the samples.
The problem is: i canīt view apmlification
any1 help me

TX

#2 pcrman

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Posted 19 May 2004 - 05:31 PM

Do you use postitive controls such as DNA that works with your PCR. Make sure all your PCR components are working.

#3 zany

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Posted 19 May 2004 - 11:59 PM

I have the same problem!
and I changed all the components ,but it didn't make differences.
the image is to vague,I donn't know why?
can you help me ?thank you! :rolleyes:

#4 Bioteck

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Posted 20 May 2004 - 07:58 AM

I use positive and negative controll, but nothing happends

#5 il0postino

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Posted 22 May 2004 - 06:11 PM

The controls are a good idea. But it sounds a bit vague to me.

Maybe use a postive control of a very diluted plasmid with the same gene (if possible) as you are triing to amplify from the plant. To test the primer pair.

Then to test for contaminations (that you indicate might be a problem) add a very small amount of plasmid DNA to your sample (any PCR DNA-primer-pair "set" that should work for sure is OK > if you do not have the control DNA mentioned above). Try to PCR this and a control sample > the same amount of plasmid DNA just by itself (so no plant sample, just PCR mix).

This should give you an idea what the problem.

#6 alex_osu3

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Posted 24 May 2004 - 03:29 PM

Hi,

How good is your DNA? Please make sure you get good quality of genomic DNA first . I remember one year ago I tried to analyze my transgene GUS in the putative transgenic plants. Actually I already confirm the gene is there and expressed by GUS staining. But I did not get all the GUS gene amplified in my samples (8 out of 11 positive, plasmid control always works). I tried different methods, including optimize PCR conditions use different Taq. After I used kit to isolate DNA I got all of them positive, which were consistant with my GUS staining.

I routinely check my DNA by running the gel with 1 KB ladder from Invitrogen and the 1636 band is 10% of input ladder. I use 1 ug ladder each time so that the mass for 1636 band is 100 ng. After taken the picture I compare the density of my smaple to band 1636 bp using Labimage software (free edition is enough for this purpose). Based on the ratio I get the concentration for each sample and 100 ng of DNA is used for PCR . Another advantage of running gel is that you can see if there is protein contaminated. If yes treat samples with proteinase K and re-purify again. I try OD method also. But the value from OD is several times high than those from gel. I get consistant results based on checking DNA concentration on gel, confirmed by house-keeping gene.

There are many so-called, simple fast methods for isolating DNA. But they may not be suitable for your situation and most of the time they have been tested in a specific system for a long time. Please remember if you want to get quick results and your boss is no poor I suggest you go for kit first. This could save you a lot of time and trouble-shooting.

As for PCR optimaztion try hot-start and touch-down method. Hot-start usually gives you more specific results and touch-down is a time-saving method for PCR: same profile for many genes when they are designed with similar parameters.

Another hit: don't try multiplex PCR if you are a beginner at PCR due to its more complexity and always include postive control and negative control in each experiment.


Hope this help,


Alex

Edited by alex_osu3, 24 May 2004 - 03:30 PM.


#7 Bioteck

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Posted 25 May 2004 - 09:04 AM

Tx very much to ALL

#8 phdconsult

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Posted 30 May 2004 - 05:14 PM

Polyprenols and other super-charged pigments may be removed from DNA samples by a 1-step mixed bed resin treatment prior to precipitation with alcohol. Degassing is also extremely important because these pigments generate free radicals in the presence of oxygen at high temperature causing DNA strand damage.




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