31 replies to this topic

[chijing]

I am attempting to create an empty vector from a commercial plasmid pCMVSport-6 (4.4kb) with Prp insert (around 2.5kb).
Therefore i carried out a double digestion on the pCMVSport6-Prp using Not1 and Sal1, incubate at 37 degree celcius for 2 hours.
Then i run the DNA on 1% agarose gel and here i can see two band. One is the vector backbone(4.4kb) and another is the insert 2.5kb.
I cut out the 4.4kb band and performed gel extraction. After extraction i run the DNA on gel, i was able to see one 4.4kb band.

The DNA eluted in TE buffer was then religate as followed:
DNA: 10ul
T4 ligase: 1ul
Buffer: 2ul
Deionized water: 7ul
total 20ul
incubate at 16 degree celcius for 1 hours and heat inactivate it at 65 degree celcius for 10 minutes.

5ul of religated DNA was then transformed into 100ul competent E coli DH5aplha.
But i can't get any colony and i would like to know where the problem lies.
for those who know, i would appreciate it if you could help.

[T C]

Hey,

Why do you need to religate the vector, if you want to clone something else in sites which are present in the clone that you already have, just digest with those enzymes and proceed; why do you want to go a step back?

Besides, just check if Not I and Sal I are compatible.

Best,
TC

[perneseblue]

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?

[almost a doctor]

perneseblue, on Apr 7 2009, 12:16 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?

You could also fill your sticky ends with Klenow, and then follow to ligate a blunt ended vector, which should work quite nicelly.

I'm assuming you want an empty vector for some transfection controls, if not... I add to TC and perneseblue, is it really necessary?

[chijing]

Actually I need the empty vector as tranfection control.
and how can i check the compatibility of Not 1 and Sal1?

by the way thanks alot for helping.

T C, on Apr 7 2009, 02:33 PM, said:

Hey,

Why do you need to religate the vector, if you want to clone something else in sites which are present in the clone that you already have, just digest with those enzymes and proceed; why do you want to go a step back?

Besides, just check if Not I and Sal I are compatible.

Best,
TC

[chijing]

T C, on Apr 7 2009, 02:33 PM, said:

Hey,

Why do you need to religate the vector, if you want to clone something else in sites which are present in the clone that you already have, just digest with those enzymes and proceed; why do you want to go a step back?

Besides, just check if Not I and Sal I are compatible.

Best,
TC

Actually I need the empty vector as tranfection control.
and how can i check the compatibility of Not 1 and Sal1?

by the way thanks alot for helping.

[T C]

Hey,

By looking at the overhangs generated after the cleavage reaction, they should match:  A should match with T and G with C in the overhangs...its fairly simple.  Look at any standard text book and it will tell you.  

Look at this:

http://bio.freelogy....ction_Digestion

Once you know how to do this, use standard tables freely available on the net to know about the compatibility in a matter of seconds.  Also, some enzymes are Isoschizomers.

BTW Not  and Sal I, as perneseblue rightly points our, are not compatible

Best,
TC

chijing, on Apr 8 2009, 09:09 AM, said:

Actually I need the empty vector as tranfection control.
and how can i check the compatibility of Not 1 and Sal1?

by the way thanks alot for helping.

[chijing]

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?

[perneseblue]

chijing, on Apr 7 2009, 08:14 PM, 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?

Yes, you do.

[T C]

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.

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?

[chijing]

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.

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?

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

[T C]

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.^^

[perneseblue]

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'

[chijing]

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, 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.^^

[T C]

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.