Troubleshooting of basic PCR - (Oct/18/2005 )
Hi all,
Last time I did PCR for my bacteria... Observation under UV only showed the marker bands and no bands were formed for my samples but the wells did glow.
Wht could have been the problem?
no DNAin the sample or no amplification...
The glow in the wells probably is in the chromosomal DNA from the bacteria that you loaded so big it gets stuck in the well.
Also, there might have been DNA on the tips u used to load your gel (in case you're using refill-package tips).
hi
u have not mentioned abourt ur reagents or PCR conditions, without that how can we suggest anything, wht was Tm of primers and wht annealing temp and time u have used. Annealing temp and time is the most crucial condition for PCR.
good luck
prabhanshu
quote name='~Js~' date='Oct 18 2005, 07:18 AM' post='28206']
Hi all,
Last time I did PCR for my bacteria... Observation under UV only showed the marker bands and no bands were formed for my samples but the wells did glow.
Wht could have been the problem?
[/quote]
When you say you saw no bands, but the wells glowed, do you mean your sample lanes showed no evidence of DNA at all on the gel itself? Not even primers?
If that's the case, everything is suspect -- PCR conditions, component quality, primer design, template quality, experimental procedure...
Thx all
here are more information on the PCR I ran...
1st denaturing= 95 C, 5mins
denaturing= 94 C, 1min
anneal= 55 C, 1mins
extend= 74 C, 1 min
final extension= 72 C 10 mins
Reaction volume(Total of 25uL):
Ultra pure H2O= 10.5 uL
2X Master mix= 12.5uL
Upstream Primer= 1uL (1pmole/1uL) Tm: 62.9 C
Downstream Primer= 1uL (1pmole/1uL) Tm: 62.7 C
*10uL of Reaction volume was added to the sample wells
As for the template, I took 1ml from my overnight enrichment broth to make the pellet.
How much template did you add?
Another thing, will it work if I resuspend the pellet in ultra pure water instead of TE buffer?
I'd guess that dH2O would actually be preferable to TE, as the EDTA will remove the Mg2+ from your reactions...
I usually just take a barely visible sample of a colony (just touch a 20 ul pipette tip to the colony) and add it to the mix.
What master mix are you using? We use Invitrogen's PCR supermixes, but have found that they seem to have titered the amount of Taq down a bit low -- so we add a smidge (0.25 ul per 100 ul rxn) more Taq.
How many cycles did you run?