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Vector insert ratio in cloning

vector insert ratio

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3 replies to this topic

#1 harry348

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Posted 07 March 2013 - 12:50 AM

Hello All,

I have a doubt abt concentration of vector and insert during cloning.
Does 5ng/ul concentartion of vector and 40 ng/ul of insert works in 1:20 ration(vector :insert ratio)??????
These low concentrations are after gel purification and doing restriction digestion.

Thanking you all in advance

Harry

#2 GNANA

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Posted 07 March 2013 - 07:44 AM

http://www.protocol-... +vector +ratio

go through the above discussion, you will get the answer for your question...
I would prefer being perfectionist rather than a passionist in Research.

I always had an alternate hypothesis....

#3 doxorubicin

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Posted 07 March 2013 - 01:21 PM

I didn't see much detail in the above link, but maybe I didn't look too carefully. Your DNA ligase will have a protocol with it. It will likely recommend a certain molar ratio of vector to insert. If you divide the concentrations by the number of base pairs for each, then your ratio will be equivalent to the molar ratio.

That is: [(5ng/ul)/200bp] : [(40ng/ul)/5000bp] will be the molar ratio if your vector is 200bp and your insert is 5000bp, for example.

#4 harry348

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Posted 07 March 2013 - 02:20 PM

thanx doxorubicin...for your reply...... my insert size is 2.4kb and vector size is 5.4kb after doing molar ratio calculation for cloning for 1:20 ratio (vector to insert) based on the above said concentration i was in doubt whether 5ng/ul of vector will be sufficient for a normal cloning reaction





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