Login

Join for Free!
118255 members


electroporation

Genetics as it applies to evolution, molecular biology, and medical aspects.

Moderator: BioTeam

electroporation

Postby ZEGSUS » Sun Oct 23, 2011 7:19 pm

I want to make electroporation but I donit know from which microbе to extract DNA and to which to insert DNA.
Which combination is the most easy?
ZEGSUS
Death Adder
Death Adder
 
Posts: 51
Joined: Wed Apr 06, 2011 10:42 am

Postby JackBean » Sun Oct 23, 2011 7:32 pm

E.coli -> E.coli

but I don't see, why would you do that just to do that.
http://www.biolib.cz/en/main/

Cis or trans? That's what matters.
User avatar
JackBean
Inland Taipan
Inland Taipan
 
Posts: 5678
Joined: Mon Sep 14, 2009 7:12 pm

Postby ZEGSUS » Mon Oct 24, 2011 5:28 am

And is it possible bacillus bulgaricus -->e.coli?
ZEGSUS
Death Adder
Death Adder
 
Posts: 51
Joined: Wed Apr 06, 2011 10:42 am


Postby JackBean » Mon Oct 24, 2011 5:43 am

sure, you can do salmon's DNA into acetobacter. It's just questionable, whether will the DNA stay or will be degraded or what you want to do with it (but I guess nothing since you don't care about the organisms :-D ).
http://www.biolib.cz/en/main/

Cis or trans? That's what matters.
User avatar
JackBean
Inland Taipan
Inland Taipan
 
Posts: 5678
Joined: Mon Sep 14, 2009 7:12 pm

Postby ZEGSUS » Mon Oct 24, 2011 6:09 am

Well as you know I want to extract DNA from bulgaricus and to insert it in E.coli.The DNA will be in ethanol in the fridge.
ZEGSUS
Death Adder
Death Adder
 
Posts: 51
Joined: Wed Apr 06, 2011 10:42 am

Postby JackBean » Mon Oct 24, 2011 7:22 am

yeah, I understood that :-D But what DNA will it be? Plasmid or gDNA?

But I guess you want to clone only small part and insert into some plasmid and express?
http://www.biolib.cz/en/main/

Cis or trans? That's what matters.
User avatar
JackBean
Inland Taipan
Inland Taipan
 
Posts: 5678
Joined: Mon Sep 14, 2009 7:12 pm

Postby ZEGSUS » Mon Oct 24, 2011 8:12 am

I will use and the both type because I haven't equipment to separate them.No , I will not clone them , I will try directly to insert the DNA into the E.coli.
ZEGSUS
Death Adder
Death Adder
 
Posts: 51
Joined: Wed Apr 06, 2011 10:42 am

Postby JackBean » Mon Oct 24, 2011 9:00 am

you have tuo purify the DNA first and that's were you differentiate between gDNA and plasmid. However, plasmid may be incompatible because of other replication origin. Inserting gDNA doesn't make sense since bacteria cannot have two chromosomes and thus would one repel probably (even keeping the plasmid without antibiotic selection will be hard).
http://www.biolib.cz/en/main/

Cis or trans? That's what matters.
User avatar
JackBean
Inland Taipan
Inland Taipan
 
Posts: 5678
Joined: Mon Sep 14, 2009 7:12 pm

Postby ZEGSUS » Mon Oct 24, 2011 9:14 am

And how to insert gDNA into the chromosome?
ZEGSUS
Death Adder
Death Adder
 
Posts: 51
Joined: Wed Apr 06, 2011 10:42 am

Postby JackBean » Mon Oct 24, 2011 10:08 am

you could use BAC (bacterial artificial chromosome), which has high capacity, but still I'm not sure, whether will it fit whole genome of your bacteria.
You want to create gDNA library?
http://www.biolib.cz/en/main/

Cis or trans? That's what matters.
User avatar
JackBean
Inland Taipan
Inland Taipan
 
Posts: 5678
Joined: Mon Sep 14, 2009 7:12 pm

Postby ZEGSUS » Mon Oct 24, 2011 11:02 am

And how to use BAC?
ZEGSUS
Death Adder
Death Adder
 
Posts: 51
Joined: Wed Apr 06, 2011 10:42 am

Postby JackBean » Mon Oct 24, 2011 11:35 am

I have never worked with BAC, but I guess cut with RE, insert your DNA and transform. Although I don't know whether electroporation will work for such a big piece.
http://www.biolib.cz/en/main/

Cis or trans? That's what matters.
User avatar
JackBean
Inland Taipan
Inland Taipan
 
Posts: 5678
Joined: Mon Sep 14, 2009 7:12 pm

Next

Return to Genetics

Who is online

Users browsing this forum: No registered users and 1 guest