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Phusion 2x not working


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4 replies to this topic

#1 Nebraskagirl

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Posted 16 June 2015 - 03:29 PM

Can this stuff go bad?  I made my genomic DNA too concentrated so I've been trying to dilute it.  I finally got a band by using 0.5ul.  However, I ran into some issue and had to go back to my diluted DNA.  I've tried several times with different dilutions but no band.  Did the phusion stop working?



#2 Curtis

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Posted 16 June 2015 - 11:41 PM

When talking about DNA it's better to mention weight rather than volume. This way the reader understands the question easier and it sounds more scientific. Phusion or its mastermix don't go bad easily. It could be the template or the primers. Phusion usually requires a higher annealing temp. I hope you are aware of that. Do you see smear?



#3 Nebraskagirl

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Posted 20 June 2015 - 04:13 PM

No I don't see anything.  I remade my primers but so far I don't see any bands.



#4 phage434

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Posted 20 June 2015 - 05:12 PM

When you have problems like this, control reactions are valuable, since they can answer questions such as "is this reagent still working?"  Without them, you have no way of knowing if your  problems lie with the reagents or other problems, such as primer quality, primer design, template quality, template amounts, cycling conditions, etc.



#5 Nebraskagirl

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Posted 21 June 2015 - 01:32 PM

I realized that the other day so I'm using some GoTaq master mix 2x to see if I figure out what is going on.  Hopefully that works.






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