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Creating empty vector by self-ligation - (Apr/06/2009 )

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T C on Apr 9 2009, 11:54 AM said:

How about this:

Find a site at the end of the gene that is already cloned in the vector or at the beginning of it , which is also present in the MCS of the vector, just splice the entire region out using this site and religate. That should work......but never tried it. :lol:

Best,
TC

chijing on Apr 8 2009, 10:44 AM said:

perneseblue on Apr 7 2009, 07:16 PM said:

Not I and Sal I are not compatible. The ligation will not work. You will have to revise the strategy. Ligating an oligo containing ends which are compatible to SalI and NotI would solve the problem.

TC does raise a point. Is creation of a blank plasmid really necessary?


Do i have to buy oligo with ends compatible to Sal 1 and Not 1 in order to do this?




Hmm..actually i'm thinking of using xho1 to replace Not1 for cutting the insert out.
Restriction site for xho1 is quite near to the restriction site for Not1, only few bases away.
and most importantly, xho1 will create overhang compatible to Sal 1.
so theoretically it'll be easier for the religation to happen since there are sticky ends at both ends of the cut vector backbone.
Am i right about this? :P

thanks a lot for helping. Truly appreciate it.^^

-chijing-

Yeah this should work....just make sure that there are no multiple Xho I or Sal I sites in yr construct.....this would complicate things.

Xho I should ideally be before Not I and in the MCS and therefore should be unique but just check. :lol:

Best,
TC

chijing on Apr 9 2009, 10:56 AM said:

Hmm..actually i'm thinking of using xho1 to replace Not1 for cutting the insert out.
Restriction site for xho1 is quite near to the restriction site for Not1, only few bases away.
and most importantly, xho1 will create overhang compatible to Sal 1.
so theoretically it'll be easier for the religation to happen since there are sticky ends at both ends of the cut vector backbone.
Am i right about this? :P

thanks a lot for helping. Truly appreciate it.^^

-T C-

or you could buy two oligos listed below, anneal them together and ligate them into a notI-SalI cleaved vector.

5'-TCGA AGATCT-3'

5'-GGCC AGATCT-3'

In this example I used BglII restriction site. You can substitute the BglII restriction site with any palindromic sequence. In this example I have also killed the NotI site and SalI site. You can easily designed the oligoes to keep those sites.

5'-TCGA C AGATCT CG-3'

5'-GGCC GC AGATCT G-3'

-perneseblue-

I have already checked..and Xho1 restriction site is before Not1 adn it's unique. I did a double digestion for my pCMVSport6-insert template using the xho1 and Sal1. Things turned out well. I saw my 4.4kb vector backbone band and have it cut out to proceed for gel extraction. After gel extraction, i run another gel to confirm presence of my 4.4kb empty vector.
Then, i did ligation and transformation into competent cells DH5alpha. After overnight incubation, i have only one single colony on my plate. so i just picked it, grow in broth and did plasmid extraction.

But it turned out that the plasmid extracted is of size between 1500-2000bp. So i assume it's probably not the 4.4kb empty vector that i wanted. Can anyone help me with this? I really have no clue what had happened. :wacko:

T C on Apr 9 2009, 01:53 PM said:

Yeah this should work....just make sure that there are no multiple Xho I or Sal I sites in yr construct.....this would complicate things.

Xho I should ideally be before Not I and in the MCS and therefore should be unique but just check. ;)

Best,
TC

chijing on Apr 9 2009, 10:56 AM said:

Hmm..actually i'm thinking of using xho1 to replace Not1 for cutting the insert out.
Restriction site for xho1 is quite near to the restriction site for Not1, only few bases away.
and most importantly, xho1 will create overhang compatible to Sal 1.
so theoretically it'll be easier for the religation to happen since there are sticky ends at both ends of the cut vector backbone.
Am i right about this? <_<

thanks a lot for helping. Truly appreciate it.^^

-chijing-

Hey,

The idea is okay, both the sites are in the MCS (chk) and you do get the vector of the right size...So the problem is with ligation or transformation. I would suggest that you repeat it again and check. Ideally you should get a lot of colonies and not just one since its religation. Hope you are using the right antibiotic and yr comp cells are fine.

Best,
TC

chijing on Apr 15 2009, 09:50 AM said:

I have already checked..and Xho1 restriction site is before Not1 adn it's unique. I did a double digestion for my pCMVSport6-insert template using the xho1 and Sal1. Things turned out well. I saw my 4.4kb vector backbone band and have it cut out to proceed for gel extraction. After gel extraction, i run another gel to confirm presence of my 4.4kb empty vector.
Then, i did ligation and transformation into competent cells DH5alpha. After overnight incubation, i have only one single colony on my plate. so i just picked it, grow in broth and did plasmid extraction.

But it turned out that the plasmid extracted is of size between 1500-2000bp. So i assume it's probably not the 4.4kb empty vector that i wanted. Can anyone help me with this? I really have no clue what had happened. <_<

-T C-

option 1. XhoI + SalI, purify backbone, then re-ligate

option 2. BsrGI (two BsrGI sites on attB sites), purify backbone, then re-ligate

-Functional Screens-

i think i'm using the right antibiotic which is ampicillin. My vector backbone has this ampicillin resistance gene.And my competent cells are fine as there is a plate full of colonies for the control.

T C on Apr 15 2009, 12:00 PM said:

Hey,

The idea is okay, both the sites are in the MCS (chk) and you do get the vector of the right size...So the problem is with ligation or transformation. I would suggest that you repeat it again and check. Ideally you should get a lot of colonies and not just one since its religation. Hope you are using the right antibiotic and yr comp cells are fine.

Best,
TC

chijing on Apr 15 2009, 09:50 AM said:

I have already checked..and Xho1 restriction site is before Not1 adn it's unique. I did a double digestion for my pCMVSport6-insert template using the xho1 and Sal1. Things turned out well. I saw my 4.4kb vector backbone band and have it cut out to proceed for gel extraction. After gel extraction, i run another gel to confirm presence of my 4.4kb empty vector.
Then, i did ligation and transformation into competent cells DH5alpha. After overnight incubation, i have only one single colony on my plate. so i just picked it, grow in broth and did plasmid extraction.

But it turned out that the plasmid extracted is of size between 1500-2000bp. So i assume it's probably not the 4.4kb empty vector that i wanted. Can anyone help me with this? I really have no clue what had happened. <_<

-chijing-

chijing on Apr 14 2009, 11:20 PM said:

I have already checked..and Xho1 restriction site is before Not1 adn it's unique. I did a double digestion for my pCMVSport6-insert template using the xho1 and Sal1. Things turned out well. I saw my 4.4kb vector backbone band and have it cut out to proceed for gel extraction. After gel extraction, i run another gel to confirm presence of my 4.4kb empty vector.
Then, i did ligation and transformation into competent cells DH5alpha. After overnight incubation, i have only one single colony on my plate. so i just picked it, grow in broth and did plasmid extraction.

But it turned out that the plasmid extracted is of size between 1500-2000bp. So i assume it's probably not the 4.4kb empty vector that i wanted. Can anyone help me with this? I really have no clue what had happened. :(


Did you digest this plasmid before running it out on the gel? If not, your size estimate is wrong, because you're comparing circular DNA to linear standards, and you might have what you're looking for already.

-HomeBrew-

Hey,

In that case, just repeat it once.

Best,
TC

chijing on Apr 15 2009, 11:36 AM said:

i think i'm using the right antibiotic which is ampicillin. My vector backbone has this ampicillin resistance gene.And my competent cells are fine as there is a plate full of colonies for the control.

-T C-

HomeBrew on Apr 16 2009, 10:41 AM said:

chijing on Apr 14 2009, 11:20 PM said:

I have already checked..and Xho1 restriction site is before Not1 adn it's unique. I did a double digestion for my pCMVSport6-insert template using the xho1 and Sal1. Things turned out well. I saw my 4.4kb vector backbone band and have it cut out to proceed for gel extraction. After gel extraction, i run another gel to confirm presence of my 4.4kb empty vector.
Then, i did ligation and transformation into competent cells DH5alpha. After overnight incubation, i have only one single colony on my plate. so i just picked it, grow in broth and did plasmid extraction.

But it turned out that the plasmid extracted is of size between 1500-2000bp. So i assume it's probably not the 4.4kb empty vector that i wanted. Can anyone help me with this? I really have no clue what had happened. :(


Did you digest this plasmid before running it out on the gel? If not, your size estimate is wrong, because you're comparing circular DNA to linear standards, and you might have what you're looking for already.


Uup..i didn't digest it..will there be lot differences between the size of a circular plasmid and a linear one?

-chijing-
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