I'm trying to amplify the whole MMP9 open reading frame (2124bp) and so far i'm having no luck. My Tm is 64 degrees and I've ran a pcr at this temp, at 60 degrees and 55 degrees and I am getting no amplification at all. My pcr cycle is: 5 mins at 94 deg then 40 cycles of 94 deg for 1min, annealing at 1 min (different temps used), 72 deg for 2 mins then a final elongation at 72 deg for 7 minutes. Each time I have done it I have got 2 non-specific bands, always at approx. 800 and 1000bp. I've tried increasing the Mg concentration, adding DMSO, increasing primer conc and increase the template but still no luck. At this point I've been told that I should go back and redesign my primers but since this is only for an honours project my budget doesn't allow it.
Is there anything else I can try?
I've read about boiling the reaction (minus taq man) for a couple of minutes before doing the PCR but don't know what this does. Can anyone help?
Thanks
I'd do a couple things: Check NEB Tm calculator (http://tmcalculator.neb.com/#!/) to find the specific annealing temp for your primers (annealing is primer specific). I don't know when you got non-specific bands, but they normally indicate an annealing temp that is too low.
Also, check the primers!. If you have the wrong primers, no matter what you change, the reaction will not work.
Third, check your polymerase. Maybe run a control to make sure it's working. See it's rate of transcription, most Taq is about 1000bp per minute, so you may need to increase the elongation time a little since your product is so large.
Here are some more resources:
https://www.neb.com/tools-and-resources/interactive-tools
https://www.neb.com/tools-and-resources/usage-guidelines/guidelines-for-pcr-optimization-with-taq-dna-polymerase
try some different polymerase. like KOD is good performer when the model is complex and fragment is long.
are you amplifying from genomic DNA or cDNA.
If its cDNA the problem is due to less efficient RT Usually Reverse transcription will not be generating your full length cDNA most often
if genomic DNA TRY SOME LONG POLYMERASE ESPECIALLY FROM QIAGEN
Ellie, long polymerases work fine for long amplicon above 2kb. I want to know what is the Tm for both forward and reverse primers and different annealing temperature that you have used?!. Moreover, your extension time (2 min) is low. It is recommended to use 1 min/kb, so you could use 2 minutes and half for extension at 72. Moreover, 40 cycles is very high numbers try to use 30-33 cycles. Hope this can help you
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