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Question about Molar ratio

infusion HD kit large insert

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4 replies to this topic

#1 Sank

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Posted 19 October 2011 - 09:28 AM

Hi everyone
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.

Regards
Sankella

#2 allynspear

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Posted 19 October 2011 - 01:16 PM

Nope, regardless of the size, the insert is still the insert. It is treated this way because a vector could potentially ligate to itself and still replicate, whereas an insert has no origin of replication or antibiotic resistance gene, so there is no way it can be replicate under selection. The standard range of 1:1 to 1:3 being optimal still applies. If you provide a more detailed description of your ligation conditions, we can possibly troubleshoot further.

Best of Luck.

#3 Sank

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Posted 20 October 2011 - 06:18 AM

Thanks Allynspear
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.

#4 tfitzwater

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Posted 20 October 2011 - 06:24 AM

The idea of molar ratio is based on the fact that you want an equal number of ends of the two (or more) DNA fragments, regardless of whether one is the vector. The old 3:1 ratio (for ng of DNA fragments) was developed when most inserts were 1 kb and the vector was around 3 kb.
For molar ratio, you want a 1:1 mix of DNA fragments.

#5 allynspear

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Posted 20 October 2011 - 10:28 AM

tfitzwater, I hate to directly disagree with you . . . but I am going to have to disagree with you. The 3:1 ratio was NEVER based on ng, it has always been a Molar ratio, which is by definition the ratio of number of molecules. The higher number of molecules of insert is based on the fact that at equimolar ratios, two molecules of vector are as likely to come together as are a molecule of vector and a molecule of insert. At a higher insert to vector ratio, the likelihood of a vector and insert interacting begins to outweigh the likelihood of vector:vector, until you reach in inflection point where insert:insert interaction begins to negatively interfere with insert:vector. This continuum is typically between 1:1 and 1:6, with 1:3 typically being optimal, although there are some differences based on type of end, size of fragments, etc.

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.





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