Posted 19 October 2011 - 09:28 AM
I have amplified a 16kb PCR fragment using primers based on Clontech infusion primer design.
Both the fwd and reverse primers have a 15bp overlap of pGL3-Basic vector (4.8 kb) with Hind III site.
The problem I have is with ligation. I have performed the cloning reaction with different ratios like
vector: insert- 1:1, 1:2, 1:3 and 1:5. When I run 1/4th of the reaction on a gel, I see two bands.
It just occurred to me that as my insert is very large do I use the standard molar calculator or should I flip it around?
May be I should consider the 5kb vector as insert & 16kb insert to be the vector.
Any help will be greatly appreciated.
Posted 19 October 2011 - 01:16 PM
Best of Luck.
Posted 20 October 2011 - 06:18 AM
I am following the manufacturer protocol.
I have tried 1:1, 1:2, 1:3 and 1:5 ratios so far.
I incubate the vector and insert with the 5X infusion enzyme mix for 15 min at 50C.
Then I use 2.5 ul out of 10ul for transformation.
Posted 20 October 2011 - 06:24 AM
For molar ratio, you want a 1:1 mix of DNA fragments.
Posted 20 October 2011 - 10:28 AM
I have even done this experiment empirically with multiple lab groups in a classroom setting where they vary the ratio of insert:vector and count transformants. With some experimental variation, 1:3 always wins out.
If you have any reference that proves me wrong, I would gladly read it.