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MOLECULAR CLONING

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MOLECULAR CLONING

HELLO EVERYONE
I am about to start my cloning and I would like to know a proper calculation for the ligation (Insert to vector). I saw many calculation but could not understand that , I would like to know how do you calculate the volume needed for the insert and the vector.. I am really troubling witrh this as its a new topic for me, I would kindly request you all to help me in this context..Eagerly waiting for the reply
with regard
sameer
siddharthsameer
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usually the insert is in 3-times molar excess
http://www.biolib.cz/en/main/

Cis or trans? That's what matters.

JackBean
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Re:

JackBean wrote:usually the insert is in 3-times molar excess

thanks for the reply but i want to undertand the complete calculation to find out the volume for insert and vector.. can u help me in that context?
siddharthsameer
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Posts: 22
Joined: Sun Jul 15, 2012 6:36 am

let's say your insert is 1 kbp long and your vector is 3 kbp long. Since you will have probably concentration in something like ng/ul, you will need to add equal weight amount of insert and vector (1 ng of 1 kbp is in mol 3-times more than 3 kbp).
http://www.biolib.cz/en/main/

Cis or trans? That's what matters.

JackBean
Inland Taipan

Posts: 5694
Joined: Mon Sep 14, 2009 7:12 pm

Re: MOLECULAR CLONING

Hi Sameer,
The ratio between insert and vector changes whether you have an cohesive (sticky) end insert or a blunt end insert. Generally while we're doing blunt end insert-vector ligation we can make up to 100:1 ratio but while we're doing cohesive end insert-vector ligation generally we prefer 1:1, 3:1 and 5:1 ratios. These ratios change with your insert size and vector size also. Here is the formula for cohesive end 1:1 ligation; ?ng insert= insert size(base pair)/vector size(base pair)x50 ng vector. I generally use 50 ng vector for ligations. You can use up to 100. So after this calculation you will find the insert amount in nanograms.
And here is a link where you can use ligation calculator
http://www.promega.com/techserv/tools/b ... calc06.htm
Good luck.
genetherapy
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thansk a lot
siddharthsameer
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Posts: 22
Joined: Sun Jul 15, 2012 6:36 am

Hello, I have a related question. I try to ligate annealed oligos into double digested (different enzymes) plasmid. The oligos I annealed were not modified with phosphates. Do I need to phosphorylate or dephosphorylate any of the two?

As far as I understand, it is not necessary to dephosphorylate my double-digested open vector as long as it was cut with two different enzymes. Thus, the 5'-end of my vector has a phosphate. But still, should I phosphorylate my insert, since its 5'-end does not have a phosphate...?
kk

Posts: 66
Joined: Fri Sep 23, 2005 7:58 pm

The purpose of dephosphorylation is to prevent self-ligation of empty vector. So unless your insert is very long, it should not be necessary.
http://www.biolib.cz/en/main/

Cis or trans? That's what matters.

JackBean
Inland Taipan

Posts: 5694
Joined: Mon Sep 14, 2009 7:12 pm

Thanks for the reply. I understand that dephosphorylation would prevents self-ligation. My problem was that whether annealed double stranded oligos need to be modified in any way before the ligation reaction. And yes, they need a 5'-phosphate added, by polynucleotide kinase, so the ligase can actually generate the covalent link.
kk

Posts: 66
Joined: Fri Sep 23, 2005 7:58 pm

Not to mention, that synthesized oligos have 5'-OH and 3'-phosphate ends... DNA ligase removes 3'-phosphates and replaces it with -OH. Thus, synthesized oligos without polynucleotide kinase treatment will participate in the ligation reaction with both their ends having -OH groups. The 3'-OH end of the oligo will form covalent bond with the 5'-phosphate end of the restriction enzyme-digested vector end, but the 5'-OH end of the oligo will have a nick next to the 3'-OH end of the restriction digested vector. Nevertheless, those nicks are fine, plasmides will be taken up by competent cells and colonies will grow.

Any comment on this? Will those nick be gone and will bacteria amplify intact circular dsDNA plasmids?

tx
kk

Posts: 66
Joined: Fri Sep 23, 2005 7:58 pm

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