Search found 14 matches

by TaintedCherub
Mon Nov 09, 2009 8:57 pm
Forum: Molecular Biology
Topic: Research in plants producing human antibodies!!?
Replies: 7
Views: 7485

I guess you would have to determine the amino acid sequence of the antibody, translate that into genetic code and synthesise the gene sequence de novo, then clone your synthetic gene into an expression vector and transform it into your plants (several ways to do this). The bad news - a lot of this i...
by TaintedCherub
Mon Nov 09, 2009 8:37 pm
Forum: Molecular Biology
Topic: NA concentration estimation
Replies: 2
Views: 2055

It can be a few things. I found it quite hard to get full information on, but I remember reading that a 260/230 peak can represent phenol (as phenolate ion), guanidium hydrochloride and some other salts. Carbohydrate contamination also affects this ratio - glycogen coprecipitate can show a 230nm pea...
by TaintedCherub
Fri Nov 06, 2009 6:18 pm
Forum: Molecular Biology
Topic: Serious cloning issues - gel extractions?
Replies: 23
Views: 28580

Re: Serious cloning issues - gel extractions?

I have done that. I got a single band, looks good. Then I gel extracted and ended up with no DNA. Anyway, doesn't matter; I had a meeting with my supervisor, we've shifted priorities and I will have to abandon this. Pity as I think I was getting close, but I really don't have time to be messing abou...
by TaintedCherub
Mon Nov 02, 2009 12:21 pm
Forum: Molecular Biology
Topic: Serious cloning issues - gel extractions?
Replies: 23
Views: 28580

Re: Serious cloning issues - gel extractions?

No, can't be helping. I did some fresh vector preps, and still seem to have too many bands... I could justify three bands by supercoiled, linear and circular, but I have four. At least they're in different places. Maybe this is progress? The six preps come from three single colonies. Image below. m
by TaintedCherub
Fri Oct 30, 2009 7:39 pm
Forum: Molecular Biology
Topic: Serious cloning issues - gel extractions?
Replies: 23
Views: 28580

Re:

I by myself don't like phenol:chloroform. Too much work, smell and not sure recovery I prefer the termo inactivation Will inactivated BamHI and CIAP be a problem for the ligation reaction? I seem to be recovering a good amount from the phenol:chloroform extractions. My advisor showed me some tricks...
by TaintedCherub
Thu Oct 22, 2009 12:01 pm
Forum: Molecular Biology
Topic: Serious cloning issues - gel extractions?
Replies: 23
Views: 28580

Re: Serious cloning issues - gel extractions?

I didn't know that! Thanks :) I will phenol:chloroform extract to be on the safe side.
by TaintedCherub
Wed Oct 21, 2009 6:36 pm
Forum: Microbiology
Topic: The largest microorganism
Replies: 3
Views: 3399

Re: The largest microorganism

These have to be in with a running! Not prokaryotic though. https://webspace.utexas.edu/lhc58/protist_slideshow/
by TaintedCherub
Wed Oct 21, 2009 5:37 pm
Forum: Molecular Biology
Topic: Spin-down speed for HEK293 after aantibody binding?
Replies: 2
Views: 6349

Re: Spin-down speed for HEK293 after aantibody binding?

I don't think you are likely to break the antibody binding by centrifugation at those sorts of speeds. I have seen reports in the literature using up to 3000 x g to pellet mammalian cells, although that seems a bit high to me. 1000 x g should be fine. If your cells are fixed you might get away with ...
by TaintedCherub
Wed Oct 21, 2009 3:46 pm
Forum: Molecular Biology
Topic: Serious cloning issues - gel extractions?
Replies: 23
Views: 28580

Re: Serious cloning issues - gel extractions?

Thanks for all your replies, and sorry for the delay in updating! I have run a couple of new gels, detailed at the bottom of this post. To try and answer some of your questions: I am using BamHI because the insert has a single BamHI site at each end, while the recipient vector is cut once in the MCS...
by TaintedCherub
Thu Oct 15, 2009 3:03 pm
Forum: Molecular Biology
Topic: Serious cloning issues - gel extractions?
Replies: 23
Views: 28580

Re: Serious cloning issues - gel extractions?

If something is carrying over from the gel extraction I guess it could be scuppering either the ligation or the transformation. Hopefully the gel should confirm whether the ligation has worked! I just started it running overnight, so should know tomorrow morning.
by TaintedCherub
Thu Oct 15, 2009 11:41 am
Forum: Molecular Biology
Topic: Serious cloning issues - gel extractions?
Replies: 23
Views: 28580

Re:

You are doing ligation at 37°C? As I remeber, I have always used like 16-18°C O/N. Did you check on gel, whether the ligation really works? Let's say, in one row have ligated product, in another unligeted mixture, in another ligated product cut with BamHI and in other ligated product cut by some ot...
by TaintedCherub
Thu Oct 15, 2009 11:34 am
Forum: Molecular Biology
Topic: Blocking o/n after adding secondary antibody (Western blot)
Replies: 3
Views: 2420

It worked fine, thanks!