Search found 11 matches

by vastgenome
Sat Mar 09, 2013 4:21 pm
Forum: Molecular Biology
Topic: Two-color in situ help
Replies: 3
Views: 4890

Problem seems to be solved. Not sure which step went wrong but the second signal began to appear when I diluted the second antibody 2X more and used less incubation time for the TSA reaction (about 3 min).
by vastgenome
Thu Feb 14, 2013 11:59 pm
Forum: Molecular Biology
Topic: Two-color in situ help
Replies: 3
Views: 4890

Thanks for the reply. I looked at whole embryos and the auto-fluorescence was so weak that it wouldn't even be picked up by our most sensitive cameras. I also suspected the endogenous peroxidase activity but that did not seem to factor in either since the anti-DIG + cy3 always worked. I thought of t...
by vastgenome
Thu Feb 14, 2013 5:00 am
Forum: Molecular Biology
Topic: Two-color in situ help
Replies: 3
Views: 4890

Two-color in situ help

Hi all, I've had this trouble for a while and couldn't figure out why. Typically I see people do in situ on slides, but I work with 96-well plates. I use the Perkin Elmer TSA plus kit (cy3/Fluorescein) for two colors. Two mRNA probes (one is Digoxigenin-labeled the other is Fluorescein labeled) were...
by vastgenome
Fri Jun 22, 2012 3:55 pm
Forum: Molecular Biology
Topic: PCR from cDNA question
Replies: 1
Views: 6205

PCR from cDNA question

Hi All, I want to PCR a 2kb long segment of a gene out of the cDNA made from total RNA extract but have been experiencing many weird problems so I hope the brilliant minds here can shed some light on it. The total RNA was extracted from ~100 whole embryos (~1000 cells each) of an invertebrate marine...
by vastgenome
Thu May 13, 2010 7:33 pm
Forum: Molecular Biology
Topic: plasmid map visualization online software
Replies: 1
Views: 1420

plasmid map visualization online software

Hi guys,

Can you recommend a good online software/ webpage based software for visualizing common plasmid vector restriction maps? I know of PlasMapper, but the functionality is somewhat limited...

thanks!
by vastgenome
Tue May 04, 2010 2:09 am
Forum: Molecular Biology
Topic: do 5'-phosphorylated primers expire?
Replies: 1
Views: 2293

do 5'-phosphorylated primers expire?

Hi guys, A naive question, as i m trying to introduce specific point mutations in the middle of a gene linked in a plasmid. The way i do it is through PCR off the entire plasmid and then reclose the circle. I ran into some problems ligating the linear PCR product, and i was wondering if the modified...
by vastgenome
Wed Apr 21, 2010 4:41 pm
Forum: Molecular Biology
Topic: signaling molecules that trigger Ras pathway?
Replies: 1
Views: 1552

signaling molecules that trigger Ras pathway?

How can i find a comprehensive list of signaling molecules that trigger the Ras pathway? I have done some pubmed search, but the papers i found are mostly focusing on the downstream pathways, i was wondering if some one can point me to the correct direct to look? thanks!
by vastgenome
Mon Apr 12, 2010 6:02 am
Forum: Molecular Biology
Topic: Error Bars--QPCR
Replies: 4
Views: 13427

Re: Error Bars--QPCR

Hi, I think a typical qPCR run would consist of at least 3 technical repeats for each unknown sample. Once you get that, the standard deviation can be calculated from these technical repeats, and can be propagated using geometric averaging rule. the error propagated in the first dCt is carried over ...
by vastgenome
Mon Apr 12, 2010 5:51 am
Forum: Molecular Biology
Topic: RNA prep help
Replies: 4
Views: 4651

Re: RNA prep help

I use RNAeasy Kits all the time and I find that the key factors are the quality of RNA and the control of contamination. Whatever you do to remove RNAses there will always be some present so I try to minimize the time between extracting RNA from it's native source and using it. If it's a tissue ext...
by vastgenome
Mon Apr 12, 2010 5:40 am
Forum: Molecular Biology
Topic: RNA prep help
Replies: 4
Views: 4651

Re:

if you're using just water for elution, you should check pH of the water. Also, I think, that let it stand for at least a while is quite crucial (did you pipette it really into the middle?). The other thing, you can do two elutions (even with the same water). BTW what volume did you use? Do you mea...
by vastgenome
Mon Apr 05, 2010 2:31 am
Forum: Molecular Biology
Topic: RNA prep help
Replies: 4
Views: 4651

RNA prep help

Hi everyone, I have been trying to prep RNA for qPCR and the yield has been extremely low even if i was very careful in handling things. I used QIAGEN's RNeasy Mini kit and the steps for RNA prep really is minimal. I followed the protocol to the letter, only taking liberty where it has been describe...