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Multiplex PCR - (Oct/17/2012 )

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hobglobin on Wed Oct 17 20:55:59 2012 said:


also in adult trout DNA?


I don't deal with adult trout DNA, i am only interested in juvenile trout. Though, the paper i referred used adult trout and they didn't have problem like me.

-Mad Researcher-

Trof on Wed Oct 17 20:58:11 2012 said:


OK, rule one. Always run negative control.
Put there just primers and reaction mix and water and see if you get faint bands there.


Ok i will run negative control tomorrow and if i face the same problem we can discuss it then.

Thanks a lot Trof.

-Mad Researcher-

By the way, the trout Y chromosome sequence is in GenBank EU081756.1 mRNA for your gene is AB626896.1
Your primers position on Y chromosome sequence are 11381 - 114033 for forward and 11701 - 11723 for reverse. The exact product length should be 343 bp and they don't seem to bind to other trout regions (though with incomplete genome this may not mean much, however there are lots of trout sequences available).

All these you could get from your paper and Blast.

-Trof-

i knew the trout Y chromosome sequence is available, i was saying the whole genome sequence of the trout is not available.

-Mad Researcher-

Trof on Wed Oct 17 20:58:11 2012 said:


OK, rule one. Always run negative control.
Put there just primers and reaction mix and water and see if you get faint bands there.


Agreed. A positive result tells you absolutely nothing with a negative negative :)

-leelee-

Decided to run a gradient PCR from 59-63 degrees with few samples. Hope that helps now.
I am also running a negative control with each sample am running.

-Mad Researcher-

Trof on Wed Oct 17 20:58:11 2012 said:


OK, rule one. Always run negative control.
Put there just primers and reaction mix and water and see if you get faint bands there.


so, i just ran a negative control but there was no band.
I have a question about negative control: if there is no DNA in that reaction then obviously there won't be any band if i run on the gel with just primers and MM. So, how do you know if the negative control has worked or not?

-Mad Researcher-

When you say you ran a negative control, do you mean you included an extra tube (with everything except template) with your other tubes in the thermocycler?

The point is that the negative control shouldn't work. This eliminates the possibility of DNA contamination in your other reactions.

For example, if the negative control had come up with a faint band (the same as your unexpected, problem band from the other reactions), then you would assume that either one of your PCR reagents is contaminated sample DNA. Or that you are accidentally transferring template over between tubes while setting your reaction up.

I hope I've explained that clearly. This is really basic stuff (not saying that to be mean, everyone has to start somewhere, we aren't born with this kinda knowledge ), so is there someone in your lab that can run through this with you?
Your supervisor really should know what level of background you have before sending you off into the lab (I'm assuming you are new to all of this?) Perhaps they assume you already know about PCR and appropriate controls? It would be worth asking them to explain from scratch (and also to do your own reading in text books etc).

-leelee-

When you say you ran a negative control, do you mean you included an extra tube (with everything except template) with your other tubes in the thermocycler? - Yes i ran it without any template.

I never run negative controls like the way you say. I use reference genes to compare. In this case, the female would serve as a negative control for me because i already knew that this sample is female and it should show only one band as compared to other samples.

-Mad Researcher-