Search found 259 matches

by biology_06er
Thu Feb 23, 2012 9:54 am
Forum: General Discussion
Topic: Inducing bacterial expression at a higher OD
Replies: 1
Views: 1223

Inducing bacterial expression at a higher OD

Hi there,

I usually induce my for protein expression when the OD of my bacteria is 0-4-0.5. Seeing as there was no expression, someone suggested I inuduce at a higher OD...such as 0.8-1..why is this...because theres more bacteria at that stage??...

Thanks
b_06er
by biology_06er
Thu Feb 23, 2012 4:21 am
Forum: General Discussion
Topic: ELISA readings after booster shot
Replies: 3
Views: 5286

ELISA readings after booster shot

Hi there, I recently did an ELISA to detect if my rabbit was producing antibodies...I got postive absorbance readings and sent away for a booster shot...again positive readings buut my question is, I diluted my antibody 1:200, 1:400, 1:800 etc etc and found that the post booster gave a lower reading...
by biology_06er
Thu Feb 16, 2012 8:18 am
Forum: Molecular Biology
Topic: fusion protein not expressing
Replies: 1
Views: 1703

fusion protein not expressing

Hi there, I recently cloned my gene of interested into a Pet32a vector containing GFP. I ran an expression test on colonies that were positive after running a single colony PCR but the before induction and after induction (sample taken every hr for 4 hrs). It didn't express so I used a new foward pr...
by biology_06er
Sun Feb 12, 2012 9:34 am
Forum: General Discussion
Topic: Making BL21 competent cells
Replies: 1
Views: 1289

Making BL21 competent cells

Hi there, I'm making competent cells for the first time and I have to use 20mM Mg2SO4 and 10mM KCl so I'm just wondering how to make these solutions. I assume I make 1M solution of each and to do so I would do the following: To make 1M Mg2SO4 I would dissolve 144.673g of Mg2SO4 in 1000mL of water bu...
by biology_06er
Mon Jan 30, 2012 8:25 pm
Forum: Cell Biology
Topic: counting cells
Replies: 3
Views: 2761

Re: counting cells

Not really...what else was I suppose to do..I diluted 2 fold there was too many cells to count..I diluted 10 fold still to many..15 fold seemed the next logical factor. I was mixing in a well before loading on to a hemocytometer so I just used useable volumes
by biology_06er
Mon Jan 30, 2012 12:52 am
Forum: Cell Biology
Topic: counting cells
Replies: 3
Views: 2761

counting cells

Hi there, The other day I had to count cells after resuspending my cells in 4mL. I took 10uL of cells mixed with 10uL of trypan blue and loaded onto a hemocytometer...I had soo many cells I couldn't count so I diluted my cells 15 fold (3.3uL of cells + 46.7uL of media +50uL of trypan blue)..I counte...
by biology_06er
Wed Jan 11, 2012 1:35 am
Forum: Molecular Biology
Topic: dot blot--protoplasts
Replies: 1
Views: 1471

dot blot--protoplasts

A question regarding protoplasts in a dot blot..my protein of interest is in the protoplast which I has isolated and I have been told to do a titration/dilution of it...so I was told do 4x 10 fold diltuin...does this mean if I started with 1:10 my next wld be 1:100, 1:1000, 1:10000/---is this right?...
by biology_06er
Fri Dec 16, 2011 9:47 am
Forum: General Discussion
Topic: HCL instead of imidazole!
Replies: 6
Views: 4251

sorry to be a pain..if I were to refold it to be on safe side..what to I refold in buffer wise/dialyze..MCAC-0 (Tris 20mM pH 8, 0.5 NaCl--the same as what I washed column with)..this causes the detanurant (Hcl) to be absorbed and hence cause protein to refold?? Have I got that right?
by biology_06er
Fri Dec 16, 2011 9:37 am
Forum: General Discussion
Topic: HCL instead of imidazole!
Replies: 6
Views: 4251

Sorry that last message is a jumble of words...so just to explain more clearly..I added 10uL of my "eluted" protein (the one I did with HCl) and added 10uL of reducing buffer-it turned yellow..I also took a sample of beads (10uL and added 10uL reducing buffer--also turned yellow)-..boiled ...
by biology_06er
Fri Dec 16, 2011 9:23 am
Forum: General Discussion
Topic: HCL instead of imidazole!
Replies: 6
Views: 4251

Re: HCL instead of imidazole!

Hi, I'm using a nickel column, when i added my reducing buffer to a sample of beads and my eluted protein it turned yellow..so i washed it with my buffer and then tested for colour change...it didn't turn yellow...so i eluted with 100mM of imadazole and ran a gel and got a band...but i had quite a b...
by biology_06er
Fri Dec 16, 2011 6:29 am
Forum: General Discussion
Topic: HCL instead of imidazole!
Replies: 6
Views: 4251

Re: HCL instead of imidazole!

or more likely will the HCl remains on the beads affect my protein
by biology_06er
Fri Dec 16, 2011 4:50 am
Forum: General Discussion
Topic: HCL instead of imidazole!
Replies: 6
Views: 4251

HCL instead of imidazole!

Hi there, Soo today I eluted with MCAC-100 (or so I thought)..I realsied once i added reducing buffer i added 1M HCL (i have them in the same size bottle next to each other on my shelf)...so what i did was i just washed my column with MCAC-0 a few times and eluted with the correct imidazole concentr...