4 replies to this topic

[js86]

hi all

i m trying to amplify my gene from sample but i am getting regular contamination.
i tried all my best now i m not getting what to do......plz any one help me...

[suryaK]

js86, on 25 June 2011 - 05:17 AM, said:

hi all

i m trying to amplify my gene from sample but i am getting regular contamination.
i tried all my best now i m not getting what to do......plz any one help me...

hi
you didn't mention what type of contamination you are getting in PCR, but try to keep these things in mind when your doing PCR
1) keep your working area clean
2) sterilized water (water vial once opened don't use for next day reaction)
3) clean pippette with ethanol before using
4) keep two reactions in two different PCR machines, if PCR contamination
5) if possible change all your working stock solutions like primers, dntps etc.,
6) if possible clean your PCR wells with DNase, followed by ethanol and water washing.

[js86]

js86, on 25 June 2011 - 05:17 AM, said:

hi all

i m trying to amplify my gene from sample but i am getting regular contamination.
i tried all my best now i m not getting what to do......plz any one help me...

thanks suryaK
i will definitely try with ur suggestions...

[nightingale]

Quote

2) sterilized water (water vial once opened don't use for next day reaction)

kindly,  i don't agree with this ...
you can just "marker" the vial you opened, and keep watching your reactions for contamination.
it is waste of money not to use a vial for more than one reaction ...
i think ...

you must write down what you are using now in ur PCR reaction,
and then start changing one item at one time, for you to know what was the source of contamination...

this is the solution, if the contamination occured while preparing the reactions.

where as for u to know if there was cross contamination between the samples in your run,
include no-template-controls between your samples.
and if one of the no-template-controls gave a positive result,
you will know that you must improve your technical skills,
as to watch out the small droplets that fall from the pipette tip from one tube to another for example.

hope my notes will be useful ...

[js86]

nightingale, on 26 June 2011 - 08:07 AM, said:

Quote

2) sterilized water (water vial once opened don't use for next day reaction)

kindly,  i don't agree with this ...
you can just "marker" the vial you opened, and keep watching your reactions for contamination.
it is waste of money not to use a vial for more than one reaction ...
i think ...

you must write down what you are using now in ur PCR reaction,
and then start changing one item at one time, for you to know what was the source of contamination...

this is the solution, if the contamination occured while preparing the reactions.

where as for u to know if there was cross contamination between the samples in your run,
include no-template-controls between your samples.
and if one of the no-template-controls gave a positive result,
you will know that you must improve your technical skills,
as to watch out the small droplets that fall from the pipette tip from one tube to another for example.

hope my notes will be useful ...

Thanks veteran for ur suggestions