Search found 259 matches

by biology_06er
Sun Dec 04, 2011 6:05 am
Forum: General Discussion
Topic: N/C terminus
Replies: 1
Views: 1477

oh and would my thioredoxin tag be on the N terminal of GFP/any gene cloned into the MCS
by biology_06er
Sun Dec 04, 2011 5:56 am
Forum: General Discussion
Topic: N/C terminus
Replies: 1
Views: 1477

N/C terminus

Hi there, I recently cloned my gene of interest into a Pet32a vector containing a GFP protein..sites BgII/EcoR1. I then fused my gene containing EcoR1/Xho1 sites. When I look at the Pet vector map, am I right in saying my gene is cloned downstream of GFP and attached to the C TERMINUS of GFP. cheers...
by biology_06er
Mon Nov 28, 2011 9:04 pm
Forum: General Discussion
Topic: Test induction-protein expression wrong dilution
Replies: 1
Views: 1075

Test induction-protein expression wrong dilution

Hi there, This morning I started a test induction and USUALLY i add 500uL of my overnight culture to 5mL of fresh media etc..this morning I somehow had 2mL fresh media in my head and added 500uL o/n culture to 2mL of fresh culture therefore the dilution factor was much less...how will this affect th...
by biology_06er
Sat Nov 26, 2011 8:31 am
Forum: General Discussion
Topic: ELISA graph plot
Replies: 2
Views: 2334

Could I just plot it as 200, 400, 800 etc and write as a 1/serum dilution (x100) (I know there a various ways as google shows but just wondering is there a standard one that works/looks best)
by biology_06er
Sat Nov 26, 2011 8:00 am
Forum: General Discussion
Topic: ELISA graph plot
Replies: 2
Views: 2334

ELISA graph plot

Hi there, I recently carried out my first ELISA, and to each well I added varying concentrations of my antibody to my protein. I started with a 1:200 dilution and then titrated 2 fold so ended up having 1:200, 1:400, 1:800, 1:1600, 1:3200, 1:6400, 1:12800, 1:25600, 1:51200, 1:102400, 1:204800, 1:409...
by biology_06er
Tue Nov 22, 2011 9:16 pm
Forum: General Discussion
Topic: Dilution-titrate 2 fold definition
Replies: 1
Views: 2921

Dilution-titrate 2 fold definition

Hi there, I have some sera and if it says "dilute sera in PBS-T (titrate 2 fold) starting at 1:200...what exactly does it mean... I'm thinking say I start with a 1:200 1mL sample...take 5uL of sera and add 995uL of PBS-T..that would be my starting diltion right?..So titrating 2 fold would mean....
by biology_06er
Sat Nov 19, 2011 10:49 am
Forum: Molecular Biology
Topic: GFP fusion protein
Replies: 1
Views: 1783

GFP fusion protein

Hi there, So I'm currently in the process of ligating my protein of interest with a Pet vector containing GFP. Once I've done that, I plan on transforming into BL21 cells, and then hopefully I will have some positive colonies..to check for +ve colonies I plan on carrying out single colony PCR...as t...
by biology_06er
Tue Nov 15, 2011 8:56 am
Forum: Molecular Biology
Topic: 2ME and SDS
Replies: 2
Views: 2466

2ME and SDS

Hi there, So I've recently been tryed to purify my protein and everytime I ran a SDS gel, I had a band at 120kda and my protein is around 30kda. I tried size exlcuion, ion exchange, but that 120 band was allways there...so then it was found out I didn't add 2ME to my reducing buffer :S...and once it...
by biology_06er
Wed Nov 02, 2011 3:20 am
Forum: General Discussion
Topic: ion exchange chromatography
Replies: 1
Views: 1175

ion exchange chromatography

Hi there, I plan on doing IEC tomorrow and need to dialyze my protein to keep a negative charge...the plan is to dialyze in Tris pH 8 (pi of my protein is 6) does that sound right...I don't have to add NaCl to the buffer right (NaCl is what I'm eluting with)..I seem to have written in my book "...
by biology_06er
Sun Oct 30, 2011 2:44 am
Forum: Molecular Biology
Topic: Wrong imidazole conc
Replies: 3
Views: 3153

Wrong imidazole conc

Hi there, Recently I've been purifying a protein and the first time I did so, I used increasing concs of Imidazole--the lowest I used was 5mM and the highest 100mM and at both and also the concs. inbetween (10,20,40,80mM) protein was eluted. So the next time I purified the same protein I planned on ...
by biology_06er
Mon Oct 24, 2011 11:13 am
Forum: Molecular Biology
Topic: GFP
Replies: 7
Views: 3903

Re: GFP

Hi, I have 2 pet vectors...one containing GFP and one containing my insert.....the GFP was has a bgii and ecor1 site and my insert has a xho1/ecor1 site....the plan is to fuse my insert with GFP so im thinking if the gfp is already in a pet vector, i need to cut it out and insert it into the vector ...
by biology_06er
Mon Oct 24, 2011 9:48 am
Forum: Molecular Biology
Topic: GFP
Replies: 7
Views: 3903

Re: GFP

Hi there, Thanks for the replies...I had a relook and it seems my GFP insert already has BgII and EcroR1 inserted (as it would as it is already in the Pet vector) then i wouldnt need primers right?... I could cut the GFP insert straight from the Pet vector using BgII and EcoR1 and likewise cut my or...