TOPO TA CLONING - MOLAR RATIO - (Dec/15/2012 )

I have a question about molar ratio for TOPO TA Cloning. What happens if you add too much PCR product to your TOPO TA cloning? I've read ratios should be 1:1 or 1:3 (vector:insert). What happens to your cloning as the ratio gets higher (1:6, 1:10, etc)? People say it won't work. I'm just curious if anyone can provide a response to what is happening to the cloning reaction? Less is better than more, correct (i.e. 1:1 vs 1:6). The package insert from Invitrogen makes it seem easy if all your doing is taking 1 or 2ul from a clean pcr reaction. However, if your having to gel purify replicates of a weak pcr reaction then quantify the DNA, it's important to calculate the correct input of insert. Suggestions, comments, experiences???

Thanks

The only effective molecules present are the circular ones with a single origin. If you have a very high insert concentration, then each end of a vector strand will ligate to a different insert strand. Those molecules will never transform. You want a low concentration of vector and an equimolar amount of insert to maximize the fraction of ligated molecules that are circular. But you also want enough of the molecules. The sweet spot is about 20 ng of vector and an equimolar amount of insert.

phage 434 - I never thought about the linear plasmid ligating to two separate inserts. That makes sense as to why the higher ratios would not work. I'm using the TOPO TA Kit for sequencing. Vector is 3956bp provided at 10ng/ul concentration. We only use 0.5ul or 5ng of vector for TOPO TA Cloning in my lab, saves money. I was trying to clone in a 510bp insert. I think I was having issues because the vector:insert ratio was too high. The first time I added 16ng of insert which would have been about a 1:25 ratio. I'm pretty sure this is why it didn't work. The lab protocol just said to add 2ul of PCR product but nothing about molar ratio. I would consult my lab members but I'm in a very small lab and the other and more senior student is not very helpful. I'm going to repeat the TOPO TA Cloning as shown below.

Using a 1:3 molar ratio:
Vector - sze = 3956bp and amount = 5ng
Insert - size = 510bp and need to use 1.93ng

Question
I usually run PCR replicates and gel purify my products then quantify with nanodrop. My eluate concentrations run between 10-30ng/ul, depending on the efficiency of PCR. Do you think it would be best to make a dilution from the eluates to a concentration of 1ng/ul to use for cloning? Concentration is too high to pipette 1ng and it feels weird diluting DNA that low. I guess I'm just inexperienced.


Thanks again!