MOLECULAR CLONING

[siddharthsameer]

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]

usually the insert is in 3-times molar excess

[siddharthsameer]

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]

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).

[genetherapy]

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]

thansk a lot

[kk]

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]

The purpose of dephosphorylation is to prevent self-ligation of empty vector. So unless your insert is very long, it should not be necessary.