Search found 22 matches

by Newbie
Thu Mar 08, 2007 8:08 pm
Forum: Cell Biology
Topic: Cell motility/invasiveness assay?
Replies: 2
Views: 2159

I've heard of matrigel, although I guess I would have to order a kit for that? isn't it expensive? Aren't micro-boyden chambers used to study chemokine effects on leukocytes? I just want to test the effects of chemotherapy drugs on the cell motility and invasiveness...can boyden chambers be used for...
by Newbie
Wed Mar 07, 2007 6:37 pm
Forum: Cell Biology
Topic: Cell motility/invasiveness assay?
Replies: 2
Views: 2159

Cell motility/invasiveness assay?

Hello experts, I am doing some tissue culture work with cancer cells and was wondering if anyone knew of any quick and simple methods to measure cell motility or invasiveness? A lot of the assays for cell motility seem too time consuming. As a last resort, I suppose there are kits out there that ser...
by Newbie
Tue Oct 03, 2006 6:36 pm
Forum: Molecular Biology
Topic: incomplete transfer of protein samples for western blot
Replies: 0
Views: 1319

incomplete transfer of protein samples for western blot

Hi all, I've been having a great deal of problem transferring my proteins over to my nitrocellulose membrane for western blots. The strange thing here is that my ladder has actually completely transferred over and even passed through the membrane while my protein samples are all mostly stuck onto th...
by Newbie
Thu Jul 13, 2006 5:29 pm
Forum: Molecular Biology
Topic: Melting Curve giving fluctuating Ct values
Replies: 1
Views: 1096

Melting Curve giving fluctuating Ct values

Hi all, I am doing some QPCR experiments with SYBR Green using cDNA and my primers never seem to work properly. I re-designed it 3 times already, the first time the primers looked promising- from the melting curve there were no primer dimers but then the CT values were flutuating up and down for my ...
by Newbie
Mon Jun 12, 2006 6:11 pm
Forum: Molecular Biology
Topic: Is it ok to replate from a bacterial lawn?
Replies: 2
Views: 1644

Hi canalon,

Yea, I was a bit afraid of my low concentration of DNA. I will re-do the transformation then! Thanks.
by Newbie
Fri Jun 09, 2006 3:01 pm
Forum: Molecular Biology
Topic: Is it ok to replate from a bacterial lawn?
Replies: 2
Views: 1644

Is it ok to replate from a bacterial lawn?

Hi all, I was doing some plating on LB plates and let it grow overnight. The next day I noticed that I had a lawn of bacterial growth, probably becuase I plated the entire transformation mix and it was way too concentrated. Anyhow, I was just wondering whether it would be ok to replate from this bac...
by Newbie
Thu Jun 08, 2006 5:05 pm
Forum: Molecular Biology
Topic: Vector is 80X larger than insert, what molar ratio to use?
Replies: 9
Views: 8227

I am transforming into DH5alpha competent cells, I think the ligation mix is alright...I'm trying it with fresh buffer and ligase in a 20ul ligation mix at 22 degrees for an hour now at various molar ratios. I re-made a fresh batch of insert and vector both around 30ng/ul. If I used 1ul of both inse...
by Newbie
Mon Jun 05, 2006 3:29 pm
Forum: Molecular Biology
Topic: Vector is 80X larger than insert, what molar ratio to use?
Replies: 9
Views: 8227

O you are right! I didn't even about that yet...it would be hard to see but as of now I'm having enough trouble just getting some colonies...one step at a time I guess...
by Newbie
Mon Jun 05, 2006 2:57 pm
Forum: Molecular Biology
Topic: smearing in bands after digestion
Replies: 3
Views: 1885

My band does migrate to the expected size but I know if I can call it clean since there is a smear...but my uncut vector doesn't have a smear though
by Newbie
Fri Jun 02, 2006 9:00 pm
Forum: Molecular Biology
Topic: smearing in bands after digestion
Replies: 3
Views: 1885

smearing in bands after digestion

Hi everyone, I was wondering why I keep seeing a smear in my bands after digestion of my vector. I digested with 1ul EcoR1 and XhoI in a 50ul reaction for 2-3 hours. I also ran the uncut vector along side it and noticed that the smear appears only after digestion. My post-doc told me to just cut the...
by Newbie
Fri Jun 02, 2006 4:42 pm
Forum: Molecular Biology
Topic: Vector is 80X larger than insert, what molar ratio to use?
Replies: 9
Views: 8227

Alright I will try it out...but if I dilute my vector I will need even less of the insert right which means less volume...but I'm trying to increase the volume of the insert while keeping the volume of the vector around 1ul...I guess I can always dilute my insert as well...we will see... :lol:
by Newbie
Wed May 31, 2006 9:21 pm
Forum: Molecular Biology
Topic: Vector is 80X larger than insert, what molar ratio to use?
Replies: 9
Views: 8227

So, using 1ul insert and vector has worked for you? Is it in a 20ul ligation reaction? Do you mind if I ask what concentration of vector and insert you start out with before and after purification?