Search found 65 matches

by SororSaudade
Fri Sep 28, 2007 3:00 pm
Forum: Molecular Biology
Topic: priciple of cloning
Replies: 5
Views: 5098

Maybe I didn't get your question... but why don't purify the fragments you want before the ligation?
Like mith said... you can also select the plasmid you want after ligation and transformation, but that's very time consuming (though you sometimes have to do it).
by SororSaudade
Wed Sep 19, 2007 3:00 pm
Forum: Molecular Biology
Topic: cloning problems
Replies: 7
Views: 4402

are you sure the restriction enzymes are working properly? I mean... If you're digesting the fragments with 2 different enzymes you shouldn't get any colony in the re-ligation control. Unless the ends are compatible... and then, your insert with that size could definitely self-ligate (hope I'm makin...
by SororSaudade
Thu Sep 13, 2007 9:43 am
Forum: Molecular Biology
Topic: How to differentiate between RNA and DNA
Replies: 12
Views: 13310

If you can spare some of you sample, I think the RNase or DNase treatment could be a very good and simple way of knowing that.
I'm not an expert though..
by SororSaudade
Wed Aug 15, 2007 10:08 am
Forum: Molecular Biology
Topic: The missing plasmid...
Replies: 4
Views: 2086

wow... thanks for the explanation :)

I already knew I have to do it all over again... just wanted to know what might have happened

Thank you once again
by SororSaudade
Tue Aug 14, 2007 8:05 pm
Forum: Molecular Biology
Topic: The missing plasmid...
Replies: 4
Views: 2086

I'm using kanamycin.

Agrobacterium are naturally resistant to rifampycin... and I also add this to the medium.

But can you explain me how do they get this spontaneus resistance?

Thanks :)
by SororSaudade
Tue Aug 14, 2007 12:24 pm
Forum: Molecular Biology
Topic: The missing plasmid...
Replies: 4
Views: 2086

The missing plasmid...

Hello all! I was trying for ages to transform some Agrobacterium cells and never manage to get any colonies. When I finally got one colony on the transformation plate I did a PCR on it and it amplified very well a fragment the size I was expecting. After this I grew these bacteria on solid and liqui...
by SororSaudade
Mon Aug 13, 2007 5:05 pm
Forum: Molecular Biology
Topic: Gel electrophoresis
Replies: 3
Views: 1877

Is just a control without DNA (or with a sequence that you absolutely know that does not amplify eith the primers you're using). It allows you to know if your mix is contaminated (it is if you get any amplification in this sample)

Any other question, just ask :)
by SororSaudade
Mon Aug 13, 2007 4:24 pm
Forum: Molecular Biology
Topic: Gel electrophoresis
Replies: 3
Views: 1877

it seems your primers also anneal in the vector alone... you can try to increase the Ta.

I supose you did like a negative control of the PCR (so that you can discard the contamination hypothesis).
by SororSaudade
Mon Aug 06, 2007 9:11 am
Forum: Molecular Biology
Topic: plasmid DNA question
Replies: 6
Views: 7467

I would transform E. coli with the plasmid + insert and then extract it with a kit, so that you can get high amounts of it very well purified. You can also store the transformed bacteria with glycerol at -70ºC and extract plasmid from them every time you want to.
by SororSaudade
Wed Aug 01, 2007 5:23 pm
Forum: Molecular Biology
Topic: article request... again
Replies: 0
Views: 1004

article request... again

Hello all once again I came across an article that would be very helpful to me, but I can't have access to it. Does somebody have it that could send me? The article is this: Wroblewski, T., Tomczak, A., and Michelmore, R. W. (2005). Optimization of Agrobacterium-mediated transient assays of gene exp...
by SororSaudade
Wed Jul 25, 2007 5:59 pm
Forum: Molecular Biology
Topic: article request
Replies: 2
Views: 1698

Oh, I'd already got it (that's why I only looked at this post now..)

But thank you anyway for your help :D
by SororSaudade
Mon Jul 23, 2007 10:50 am
Forum: Molecular Biology
Topic: article request
Replies: 2
Views: 1698

article request

Hello all!

Can anyone get this for me?

Mersereau M., Pazour G.J., Das A. 1990. Efficient transformation of Agrobacterium tumefaciens by electroporation. Gene 90: 149–151

I can't have access to it and I really need it :(

Thanks in advance!