## MOLECULAR CLONING

Discussion of all aspects of biological molecules, biochemical processes and laboratory procedures in the field.

siddharthsameer
Garter
Posts: 22
Joined: Sun Jul 15, 2012 6:36 am

### 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

JackBean
Inland Taipan
Posts: 5694
Joined: Mon Sep 14, 2009 7:12 pm
usually the insert is in 3-times molar excess
http://www.biolib.cz/en/main/

Cis or trans? That's what matters.

siddharthsameer
Garter
Posts: 22
Joined: Sun Jul 15, 2012 6:36 am

### 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?

JackBean
Inland Taipan
Posts: 5694
Joined: Mon Sep 14, 2009 7:12 pm
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.

genetherapy
Garter
Posts: 33
Joined: Fri Nov 11, 2011 9:54 am

### 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.

siddharthsameer
Garter
Posts: 22
Joined: Sun Jul 15, 2012 6:36 am
thansk a lot

kk
Posts: 71
Joined: Fri Sep 23, 2005 7:58 pm
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...?

JackBean
Inland Taipan
Posts: 5694
Joined: Mon Sep 14, 2009 7:12 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.

kk
Posts: 71
Joined: Fri Sep 23, 2005 7:58 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