Search found 14 matches

by dinofernando
Fri Aug 05, 2011 6:21 pm
Forum: Molecular Biology
Topic: An interesting PCR smearing...I dont know what to do :(
Replies: 9
Views: 13690

Re: An interesting PCR smearing...I dont know what to do :(

Have you tried double checking your program setup on your thermocycler?

Might have accidentally changed it?
by dinofernando
Thu Mar 17, 2011 6:47 pm
Forum: Molecular Biology
Topic: RNA Purification via Spin column kit
Replies: 0
Views: 1096

RNA Purification via Spin column kit

Hi everyone, I'm having trouble with extracting RNA via Qiagen spin column kit. Every time I do the procedure I get excellent yields around 1000 - 2500 ng/uL but horribly low purity 1.5 - 1.7 A260/A280. I know for pure RNA I need a ratio of ~ 2.0. I'm going nuts people. I'm growing my cells to OD of...
by dinofernando
Sat Feb 27, 2010 10:36 am
Forum: Molecular Biology
Topic: Bands on electrophoresis gel
Replies: 3
Views: 1621

Here's a question. If you have 100% digestion of vector, meaning your vector is linearized why can't RNApol synthesize protein when transformed into an e.coli cell?
by dinofernando
Tue Feb 23, 2010 12:02 pm
Forum: Molecular Biology
Topic: Buffer Considerations when using Anion Exchange (FPLC)
Replies: 2
Views: 2578

Or am I able to dialize my protein into a more basic buffer?
by dinofernando
Tue Feb 23, 2010 12:01 pm
Forum: Molecular Biology
Topic: Buffer Considerations when using Anion Exchange (FPLC)
Replies: 2
Views: 2578

Buffer Considerations when using Anion Exchange (FPLC)

Hi Everyone! Quick question about protein purification using Anion Exchange column, I'm a little new to the equiptmnt and protein purification. I have a HIS-tagged protein that has a pI above 7.5. I'm using a 1M Tris, 50mM EDTA buffer at pH 7.5. So that means in this buffer my protein will have a ne...
by dinofernando
Mon Nov 12, 2007 10:50 pm
Forum: Cell Biology
Topic: Purpose of Mannitol in Mito Isolation
Replies: 0
Views: 1691

Purpose of Mannitol in Mito Isolation

Hi everyone,

Quick question what is the purpose of Mannitol in mitochondrial isolation. I know its an osmotic diuretic so does that mean it dehydrates the mitochondrial before lysis?

Thanks
Dino
by dinofernando
Sun Sep 02, 2007 2:26 pm
Forum: Molecular Biology
Topic: analyzing a ligation before transformation
Replies: 8
Views: 15419

Well I checked out my transformants over the weekend and THEY GREW!!! But I also got growth on my controls however not as much. The colonies on my transformed plate are few and "thick". The controls are thin and many so I don't know which is good or bad??? They have been in the incubatior ...
by dinofernando
Sat Sep 01, 2007 1:32 am
Forum: Molecular Biology
Topic: analyzing a ligation before transformation
Replies: 8
Views: 15419

I think the ligase should be ok. Its used commonly among all in the lab and they seem to be using it fine.

If this third try doesn't work I'll make up enough so after ligation i can run on a gel.
by dinofernando
Fri Aug 31, 2007 4:05 pm
Forum: Molecular Biology
Topic: Need help!!molecular cloning
Replies: 11
Views: 5753

I'm using the Axygen system
by dinofernando
Fri Aug 31, 2007 2:52 am
Forum: Molecular Biology
Topic: Need help!!molecular cloning
Replies: 11
Views: 5753

Method used.

How are you purifying your plasmid, phenol chloroform or a kit. I use a kit and it works great very high yield and very fast.
by dinofernando
Fri Aug 31, 2007 2:46 am
Forum: Molecular Biology
Topic: analyzing a ligation before transformation
Replies: 8
Views: 15419

Serious Ligation issues :( (Part science part rant)

Hi everyone, Well i've started my ligations and now that i'm on my third attempt to transform my XL1 supercompetent cells I've come for HELP!!! The last two that I did were directly from a .7% nusieve gel that i washed with dH20 spun and removed supernatant (well 120uL from the top since no layer wa...
by dinofernando
Mon Aug 20, 2007 11:23 am
Forum: Molecular Biology
Topic: Removing the STOP codon to make a fusion protein.
Replies: 2
Views: 8886

I've actually got it figured out. Its the stop codon of the protein and GFP is fused to the proteins C-terminal end. When I was designing the primer I just left that part (stop codon) out of the primer and started it one codon before the stop. Then added my restriction enzyme site after that. Then m...