Hi all, I am getting a consistent band near the target size (1.4kb) at the NTC of a Phusion reaction. I have changed all my stock reagents: Phusion buffer, Phusion enzyme, dNTP, Nuclease-free water, Primer stock, pipettes, etc.
Interestingly, when I used another set of primers (a totally random primers set, not for this specific Phusion reaction) for another NTC reaction, I am not getting a band. The band only appears at the NTC of my specific Phusion primers. Can anyone help resolve this?
Gel image description:
Plasmid size: 1.4kb
Top Lane 1. : 1kb DNA ladder
Lane 2-13: 12 reactions with DNA template added
Lane 14. : 1kb DNA ladder
Bottom Lane 1: 1kb DNA ladder
Lane 2: NTC with target primers
Lane 3: NTC with non-target primers
Lane 4: 1kb DNA ladder
Hey Kyle Skalenko, Thank you for the reply!
Due to this issue: 1) I purchased brand-new primer stocks (100mM) dated 17th June 2022. 2) For every repeat of this Phusion reaction, I always dilute my stock primers to 10mM.
Both the old and new primer stocks produced a consistent band near the target size for NTC.
Hi,
how many cycles are you runnin gin your PCR? For me it looks like your targets are over amplified already, so you might reduce by 5 cycles. The NTC band might be contamination so also here using appropriate decon measures and running less cylces will help.
Good Luck!
Sven
Hey Sven Reister,
I'm already using 35 cycles for this reaction. Plus I've already made all the necessary decon steps (tips, reagents, pipettes, workstation) but the NTC band is consistently appearing for the NTC of target primers, and not for the NTC of non-target primers.
Hey Kyle Skalenko,
Yup, I am already using 35 cycles. So, not sure whether reducing the number of cycles will help. I'll try the DMSO and increase the stringency of the reaction.
Thanks!
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