4 replies to this topic

[chester_ny]

i tried to do genotyping (fly transgene in mice)
with primer sets designed from transgene.
expected product size is 200-400 bps
I use Taq

my PCR cycle is 2 min 95 degree initiation
                               30 sec 95
  40 cycle of            30 sec 58
                               1 min 72 elongation total
                         10 min 72 final elongation

i tried 20s sample , they all showed positive results (exact size bands)
it's strange
so i tried to run pcr with plasmid ,
with wildtype (wth the wildtype showed bands too! exact size !)

at final I run the PCR without template (heck , bands come as well )


I didn't do hotstart
should I go for hotstart?
or my reagent got contaminated? (so frustrated)

thanks for help

[mario2004]

Contamination should be ruled out first. I think 40 cycles are too many.

[RecklessTech]

I have the same problem going on right now! I've run every contamination check possible and it's still happening.  What are your Reaction concentrations?

[seanspotatobusiness]

Expected product size is 200-400 bp? Can't you be any more certain than that? If you use a higher percentage of agarose in your gel and run it at a lower voltage (e.g. 2% and 50-60 volts), you'll get better resolution. What about when you use water as a template?

[bob1]

I would say that your reagents are contaminated... and that probably more that one reagent is contaminated.  You should throw all your reagents and start again (though you can keep the more expensive bits such as Taq and test it for contamination) - First, get clean water!(MilliQ or equivalent, or buy bottled water), make fresh dilutions of your primers, fresh dNTPs, fresh Mg²⁺ and buffer.  Clean your pipettes well and keep a pipette separate which you only use to load gels, as this is usually where contamination comes from.