Multiplex PCR - (Oct/17/2012 )

I am running a multiplex PCR with sDY (sex determining primer) and 18s (control) primer to determine the sex of juvenile rainbow trout.

I am using 1.25 uL of PCR buffer (sigma), 250 micro molar of dNTP, 0.25 uL of Jump start Taq DNA and 0.4 microl molar of (sDY primer both F and R) and 0.1 micro molar of 18s Primer (both F and R). i take 40 ng of DNA and its a 12.5 uL reaction.

The problem i am facing is that i should get a single band around 400 bp if it is a female and double bands around 200-210 bp if it is a male. But now i am getting a faint (lighter) band which shouldn't appear in case of the female.

PCR CYCLE:

Initial denaturation at 94 degrees for 1 minute, denaturation for 60 sec at 94°C; 35 amplification cycles of 94°C for 30 sec, 60°C for 30 sec and 72°C for 30 sec; and a final extension of 3 min at 72°C

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What should i change so as to avoid the double band in case of female? I have already considered reducing the amount of DNA (to 30 ng).

Please explain the structure of the sDY assay, what does it amplify? And what is the DNA difference between male and female in that region.

The sdY gene encodes a putative protein of 192 amino acids that displays sequence homology with the car-boxy-terminal domain of interferon regulatory factor 9.
sdY gene was a male-specific genomic DNA sequence in the rainbow trout.
sDY is a master sex determine gene.

Yeah but where are primers located? Where are the homologies? On the edges? Otherwise I still don't get how it could make longer sequence in females that lack this gene, and shorter in males. It's not ARMS assay or anything, so I need to see the whole schematic of the homologous regions and primer positions. There is no way how to understand what should be amplified without that.

i messaged you a pciture of the sDY region. That is all they have mentioned. They have focoused more on In-situ hybridization.

You should have posted it here in the thread, others might be interested too..

Anyway so you don't have a sequence of your primers and you don't know where they are? How do you want to opltimise that?

The gel image at the top of your picture shows somethign different than you wrote, there is one bigger band of 18S control in both males and females, and one smaller only in males, amplifing sDY. So, primers must be specific for sDY only.
So you are getting those "male" bands in female samples?

There could be contamination (what about your negative control?) /cross-contamination or nonspecificity, but if you don't know sequence of the primers you can't find out where else they could bind. And if you do know the sequence, then you could blast them to the trout sequence and find out where they bind in the first place.
Generaly increasing the annealing can help if it's nonspecifity.

Sorry about that, i couldn't find the option here.

I have the primer sequence but i don't know where they are.

Yes, what you say about the gel image is correct. I was mistaken. As i said, the bands appear in males but also in female (faint). There is no genomic sequence available for the trout yet.

I have increased the annealing temp to 61 (the primer melting temp is 62.8 for sDY and 62.2 for 18s) but it didn't help. I am planning to lower the temp to 58 or 59 to see if it helps.

Thiose faint bands, were there from the beginning or they started to appear later? Is your negative control negative (no faint bands)?

they were present in every multiplex i ran. I don't have a negative control but i have a positive control and they appear in the poistive control as well (the faint bands)

also in adult trout DNA?