Tips for visualizing very faint bands in agarose gels? - (The PCR ain't going to get better, so the gel must!) (Apr/28/2010 )
gebirgsziege on Apr 29 2010, 04:36 AM said:
to get DNA out from). You are using a six primer multiplex....would it be possible to "split" into two multiplex PCRs, so you can modify the conditions for the primers better and improve the yield? Or is ther a possibility for a nested approach (like unspecific primers for your gene first for 5 - 15 cycles, cleanup, and then multiplexing)?
For better visualising: loading the whole 20µL on the gel might help or longer staining times. Small combs, thin gels, and not too much stain to reduce the background.
Thanks... I do feel like I've spent a lot of time (and even more money) trying to find the best extraction protocol, only to return after 2-3 months of tinkering to what I was using in the first place! My samples are not that fresh (between 2-3 years old), and they were stored in a substandard freezer in a tropical country in a house with regular power outages, so... some of it is just the nature of the beast. I may try a nitrogen tank in the future (another researcher did do this for her plant samples).
I guess I could split up the multiplex, although the six different primers seem to play nice together with regard to to positive control. I think I will try another suggestion of increasing the number of cycles. My concern with a nested approach is that I would have to amplify a fairly large chunk of the COII gene to get all of the primers in there, and I don't want to go much about 500 bp becuse the recovery does drop -- I've had very poor luck trying to amplify anything longer than 700 bp. But it is certainly worth a shot. Thanks!
Everyone else has really good suggestions for trying to optimise your results, so I can't add anything there.
But I did want to say, DON'T PHOTOSHOP your gel pic if you are going to use it for publication- its just plain dodgy!!
Good luck getting it to work 
leelee on Apr 29 2010, 07:02 PM said:
But I did want to say, DON'T PHOTOSHOP your gel pic if you are going to use it for publication- its just plain dodgy!!
Good luck getting it to work
So I can't draw the bands in that I want?
Maybe this is a silly question, but can you add the SYBR to the loading buffer and samples, and then load them on a plain gel? Maybe that could decrease your background and improve visualization of the samples.
edited for grammar.
KimWG on Apr 30 2010, 12:13 PM said:
leelee on Apr 29 2010, 07:02 PM said:
But I did want to say, DON'T PHOTOSHOP your gel pic if you are going to use it for publication- its just plain dodgy!!
Good luck getting it to work
So I can't draw the bands in that I want?
hahahahaha apparently not- go figure??
Hay
in our lab we're using SYBR safe. Recently a representative adviced GelGreen (from VWR, company in Belgium) or GelRed. I find it much more sensitive then SYBR safe and safer , doesn't penetrate the skin. Anyhow, our bands were much much brighter
Hopefully useful