33 replies to this topic

[leelee]

Trof, on 17 October 2012 - 12:58 PM, 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

[Mad researcher]

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 17 October 2012 - 12:58 PM, 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?

[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?

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).

Edited by leelee, 18 October 2012 - 06:21 AM.

[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? - 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.

[leelee]

I see what you are saying, but you should always include a negative (or if you prefer "no template") control, for the reason I stated above- to rule out contamination.

Have you used these primers successfully in the past and only now they aren't working? Was your faint band there in the samples this time (the time you ran the no template negative?)?

[Mad researcher]

leelee, on 18 October 2012 - 07:14 AM, said:

I see what you are saying, but you should always include a negative (or if you prefer "no template") control, for the reason I stated above- to rule out contamination.
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Yes i understand the part of including a negative control, there was no contamination.

Have you used these primers successfully in the past and only now they aren't working? Was your faint band there in the samples this time (the time you ran the no template negative?)?

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Yes, i have used these primers before and it was successful. But now i realize my mistake, these primers were freeze-thawed a lot of times so may be because of that it created a problem but today i prepared fresh aliquots of my stock primers and used them. And secondly, as i said earlier i ran a gradient PCR and now i know the favourable temp. for these primers are 62-63 degrees not 59-61 degrees as i was using before.

Thanks a lot for the help :-)

[leelee]

Ok cool, glad you got it sorted. Sounds like you were having a bit of non-specific binding going on in there. Hope it keeps working for you.

[Mad researcher]

I hope so too. Running another PCR with unknown DNA (i.e. Sex unknown) along with my positive and negative controls (male and female which i know).

[hobglobin]

Mad researcher, on 17 October 2012 - 01:07 PM, said:

hobglobin, on 17 October 2012 - 12:55 PM, 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.

Why I asked is, because I vaguely remember that sexes in some fishes are not that fixed and can change 8also depending on environmental conditions)...though I'm not a fish expert and have no idea how it is determined in trout, perhaps the juveniles still are not that definitive and you might get unclear results, because of this.
Anyway just a thought.

[Mad researcher]

hobglobin, on 18 October 2012 - 08:13 AM, said:

Mad researcher, on 17 October 2012 - 01:07 PM, said:

hobglobin, on 17 October 2012 - 12:55 PM, 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.

Why I asked is, because I vaguely remember that sexes in some fishes are not that fixed and can change 8also depending on environmental conditions)...though I'm not a fish expert and have no idea how it is determined in trout, perhaps the juveniles still are not that definitive and you might get unclear results, because of this.
Anyway just a thought.

Yes it is true that in some fish like Clownfish which changes sex and it is then difficult to determine (though haven't tried that). In trout it is easy to determine in mature trouts i.e. when they are 2-3 years old but in case of juvenile it is difficult to determine without opening and checking the gonads. Therefore, my project involves determining sex in juvenile trouts by taking fin clips, extracting DNA and running multiplex PCR and then identifying male and female.

[Trof]

Non-template control is a key quality management feature. Without it, you can never be sure if your results are not false positive. Also without positive control, you can't tell if the sample isn't false negative. Both controls are amplified from the same mix as other samples.

In some cases one of these has more importance than other, in case of just amplifying some part of gene for subsequent use, positive control only serves as a backtrace in case the reaction is all negative. In that case you know that something is wrong, because you expect product, but without positive control you can't tell however if the problem is in reaction itself or the template. So you have to repeat the reaction (this time with positive control included), this only costs you time and reagents, but you know you have to solve something.
Non-template control  on the other hand is essencial on almost all cases, because if you expect a product and get one, you don't suspect problem at all. That's why NTC has to be present all the time. Otherwise you could get false positives and don't know about it. Mostly this comes from the reagent contamination (to a lesser extent from crosscontamination of a single sample, which is undetectable by NTC) and NTC uses same reagents.

But, if you are detecting something with PCR, and you don't know if it should be negative or positive (detection of virus or so) you always need to include both positive and NTC control, becase you can't tell without checking, that the sample is negative for virus. That can have huge consequences in diagnostics to tell someone he's negative or positive if it's not true.

[Mad researcher]

So, i did a multiplex PCR again today but i increased the annealing temp. to 62 and decreased the no. of cycles to 32 instead of 35. As you guys suggested i also had a negative control which worked out fine. The only problem is that i still have those faint bands but this time they got very faint and unless i illuminate it a lot on the trans-illuminator it cannot be seen.

[Mad researcher]

Hello guys,

I am experiencing a new type of Problem. I was doing a multiplex again (before it was working fine) but i didn't get any bands at all.  
I thought maybe i missed something so i repeated it again but again the same results. Then i changed the primers (made new stock and aliquots) tried again but didn't work. So i decided to run sDY and 18s primers individually but still no result. The last time it showed bands was when i ran a gradient PCR for the sDY F and R primers. I have also included a NTC and also included my positive and negative control (the known male and female DNA samples)

I tried both increasing and decreasing the no. of cycles. it didn't help.

I also tried with new Taq Polymerase (jump start taq DNA polymerase - sigma) that i ordered, new PCR buffer and new stock of dNTP too.

Any idea what should i do now?

Edited by Mad researcher, 01 November 2012 - 02:42 AM.

[leelee]

Same template as last time? Or new template?

And if old template, how has your template been stored (temp, buffer, etc)?