Protocol for making a recombinant plasmid

[Mega]

I'm currently looking for a protocol to cut a plasmid and ligate the inserts into another.

(I asked a proff a while ago if I could get SalI. Now he replied 'yes'. So I'm cutting out the light gene casette of pVIB ( http://images.carolina.com/images/en_US//local/products/detail/211447_bit.jpg - only one cutting site?)  and  insert it into a kanamycin resistant plasmid.)

I won't do electrophoresis. Just plate it on  Kanamycin, then some collonies will glow, while others will not. Then I take a glowing one, grow it, and do a miniprep.


But how much RE will I need, how long to wait until cut, how much plasmid and how much DNA ligase?
Would be terrific if somebody could send me a protocoll!!

Thanks!!

[Jeswin]

On Tue, Jun 5, 2012 at 10:36 AM, Mega <masters...@gmail.com> wrote:
> (I asked a proff a while ago if I could get SalI. Now he replied 'yes'. So
> I'm cutting out the light gene casette of pVIB (
> http://images.carolina.com/images/en_US//local/products/detail/211447_bit.jpg
> - only one cutting site?)  and  insert it into a kanamycin resistant
> plasmid.)
>

You need SalI cut sites in the plasmid used for religation.

> I won't do electrophoresis. Just plate it on  Kanamycin, then some collonies
> will glow, while others will not. Then I take a glowing one, grow it, and do
> a miniprep.
>

Without GE, how do you know which segment of the cut dna you are
using? You need to purify the DNA section containing the light gene.
Could it work without purification? Maybe, but it might be harder to
do.

> But how much RE will I need, how long to wait until cut, how much plasmid
> and how much DNA ligase?

I use 1uL RE, buffer (make sure you get the buffer that SalI needs),
and BSA. I use 500ng or more plasmid, less if okay, I think. Total
volume is 30uL. You can cut for 1 hr at 37C.

For ligation, check out this site:
http://www.methods.info/Methods/RNA_DNA/ligation_simple.html

[Andreas Sturm]

>You need SalI cut sites in the plasmid used for religation.

Clearly.

>Without GE, how do you know which segment of the cut dna you are

using? You need to purify the DNA section containing the light gene.
Could it work without purification? Maybe, but it might be harder to
do.

 As I see it, there are 4 [EDIT: no, 6]  possible outputs:

Ampicillin self ligated (will be destroyed by kanamycin)
Amp Lux (original)  (destroyed)
Lux - ligated (destroyed, no ORI)

Kanamycin + Amp Ressistant
Kanam - Self ligated
Kanam + pVIB

Three of them will survive in selection medium.  1 out of three will glow. This one I'll take and streak out on another pate, ( and do a miniprep later)