electroporation

by **ZEGSUS** » Mon Oct 24, 2011 11:45 am

And what about the plasmids?

by **JackBean** » Mon Oct 24, 2011 12:09 pm

these should be fine, but I don't know whether they have compatible the replication origins

by **ZEGSUS** » Mon Oct 24, 2011 12:12 pm

So if I want to insert gDNA first I need to cut it and after that to insert it?And how to transform it?

by **JackBean** » Mon Oct 24, 2011 12:50 pm

I don't know how big is the genome, but probably yes, cut, ligate, and transform. I'm not sure what is the size limit for electroporation.

by **ZEGSUS** » Mon Oct 24, 2011 2:03 pm

The size ot the lactobacteria is 2.3 Mbp.The limit is <10Kbp.

by **JackBean** » Mon Oct 24, 2011 4:22 pm

here http://en.wikipedia.org/wiki/Bacterial_ ... chromosome they put the common size of insert into BAC is up to 350 kbp. Thus the number of clones you had to test would be N = [ln(1-P)]/[ln(1-[350/2300])] where P is the probability you will find your gene of interest or whethever are you looking for (99.5%) and 350/2300 is the ratio of insert and whole genome size. As I calculated it the N is ~4, what is odd, since the ration genome/insert is 6.5 :lol:

I guess best to contact someone who is experienced e.g. someone from some sequencing company/group.

What is your aim again?

by **ZEGSUS** » Mon Oct 24, 2011 4:47 pm

To insert DNA from the bulgaricus in E.coli.

by **ZEGSUS** » Mon Oct 24, 2011 4:53 pm

JackBean wrote:here http://en.wikipedia.org/wiki/Bacterial_ ... chromosome they put the common size of insert into BAC is up to 350 kbp. Thus the number of clones you had to test would be N = [ln(1-P)]/[ln(1-[350/2300])] where P is the probability you will find your gene of interest or whethever are you looking for (99.5%) and 350/2300 is the ratio of insert and whole genome size. As I calculated it the N is ~4, what is odd, since the ration genome/insert is 6.5

I guess best to contact someone who is experienced e.g. someone from some sequencing company/group.

What is your aim again?

So you mean to cut the gDNA and to attach it to F-plasmids and after that to insert the plasmids into E.coli?

by **JackBean** » Mon Oct 24, 2011 4:56 pm

yes, that's how BACs work

by **ZEGSUS** » Mon Oct 24, 2011 5:17 pm

And how to get F-plasmids?

by **JackBean** » Mon Oct 24, 2011 6:35 pm

sorry, I have really no idea. But I guess the material providers like Invitrogen or whatever should probably have system for making gDNA library.

by **ZEGSUS** » Mon Oct 24, 2011 6:49 pm

http://aem.asm.org/cgi/reprint/60/11/4192.pdf
Well I guess that is possible to insert "naked" DNA into bacteria.

electroporation

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