Did 16sRNA sequencing to try and identify species of CoNS. Ladder ran fine but there is a faint fuzzy line at the bottom of the gel, wondering if the DNA ran off, or maybe primer dimers? Did PCR twice, for the second one we used more colonies and also changed PCR cycles. Any idea why both times the PCR looks like the picture?
Method:
- 1µl of lysostaphin + Single colony of growth
- incubate for 15 minutes at 37º
master mix contained
- 5µl of primer 8F (5′-AGAGTTTGATCCTGGCTCAG-3’) and 1492R (5’- GGTTACCTTGTTACGACTT-3’) each
- 160µl of mango mix
25µl of master mix and 5µl of prepared DNA into PCR tube
PCR
a. 95°C for 3 minutes followed by 35 cycles of:
b. 30 secs denaturation at 95°C
c. 30 secs annealing at 45°C
d. 2 minutes elongation at 72°C.
e. Final 1 cycle of 72C for 10mins.
Revised PCR
a. 95°C for 3 minutes followed by 35 cycles of:
b. 30 secs denaturation at 95°C
c. 1 min annealing at 45°C
d. 2 minutes elongation at 72°C.
e. Final 1 cycle of 72C for 10mins.
It is clear that the PCR was unsuccessful, and the faint bands in the gel are primer dimers though. I see the ladder bands in the gel and suppose that your gene of interest is larger than the smallest band of the marker, so if the PCR was successful you could have seen the desired band on the gel. Failed PCR can be caused by different reasons you need to check them. Existence of the template DNA and its quality, PCR reagents and their quality/efficiency, primers and their efficiency, PCR conditions (temperatures and time), etc. I recommend verifying the above factors. In the meantime, don't forget to include positive and negative control samples in your experiment, this will help you get an answer as to why the PCR failed.
Is the annealing temperature already known? If not you might change the annealing temperature. Try a gradient pcr to determine it.
Hi,
I did also use the earth microbiome primer but used 55°C annealing, are you sure about your temperature profile used? I would tend to say the annealing is not working as it should
Saber Delpasand Khabbazi has good point, that the marker tells you, whether you should see your amplicon. But that smear at the bottom are most likely primers.
Anyway, what polymerase did you use? Your cycling seems a little unusual. The annealing temp is rather low (but that should not be problem), but also annealing time is long. What was your amplicon length?
Do you have anything that you could use as positive control? For beginning, even anything unrelated to your samples/primers. Any plasmid for which you have primers with similar length as your desired amplicon to confirm your PCR is working at all.
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