Search found 7 matches

by clevermizo
Wed May 26, 2010 5:10 pm
Forum: Molecular Biology
Topic: What else can I do with my PCR?
Replies: 8
Views: 3922

Re: What else can I do with my PCR?

Well it sounds like template amount is the only thing that the OP hasn't tried to optimize yet, so it's probably worth a shot.
by clevermizo
Tue May 25, 2010 9:44 pm
Forum: Molecular Biology
Topic: What else can I do with my PCR?
Replies: 8
Views: 3922

Re: What else can I do with my PCR?

I would strongly suggest varying the amount of starting template. I've used above 100 ng genomic DNA to seed PCR. 50 ng cuts it for plasmids, but with genomic DNA remember that it's not 50 ng of your target sequence, it's 50 ng of the entire genome. You need to increase your odds a little. I would j...
by clevermizo
Thu May 13, 2010 7:54 pm
Forum: Molecular Biology
Topic: Dilution question
Replies: 3
Views: 2002

Re: Dilution question

The units are arbitrary at this point and don't affect the calculation, since the starting units and the ending units are the same. If I have a solution of 18 fiddledeecups and I want to dilute it down to 2 fiddledeecups, well that's a dilution factor of 18/2 = 9 . So, if I want 9 mL, I use 1 mL of ...
by clevermizo
Sat Apr 03, 2010 3:04 am
Forum: Molecular Biology
Topic: Error Bars--QPCR
Replies: 4
Views: 13431

Re: Error Bars--QPCR

Not sure, although I don't see the point of error bars without replicate experiments. Why not just perform replicate experiments?
by clevermizo
Wed Feb 03, 2010 5:37 pm
Forum: Molecular Biology
Topic: Biology Lab Help - Protein Assays
Replies: 4
Views: 9233

Re: Biology Lab Help - Protein Assays

Finished all parts of my lab and report except this final question. I'm not quite sure I fully understand the concept of why it happens. Most proteins have an absorption maximum at 280 nm. If that is so, why do you assess the absorbance in the Bradford assay with the wavelength set at 595 nm? (2 re...
by clevermizo
Thu Jan 28, 2010 4:15 pm
Forum: Molecular Biology
Topic: TRUE postives or non specific bands, pls help
Replies: 13
Views: 5639

I wouldn't believe anything on that gel as a positive. It looks like crap. You have to optimize PCR. If the assay worked previously, look back and make sure you have the exact conditions. The most important thing in my experience when working with genomic DNA is the amount of template seed. I've use...
by clevermizo
Thu Jan 28, 2010 4:03 pm
Forum: Molecular Biology
Topic: ligation rxn on gel was a smear
Replies: 2
Views: 2379

Re: ligation rxn on gel was a smear

With that much DNA in there you probably have a lot of incorrect products. Typically I use <100 ng total DNA (i.e., 50 ng vector and 3:1 molar excess of insert). I can't really recommend using 1 ug of DNA in a ligation reaction. Now, if you use as small of an amount of DNA as I do, you won't likely ...