4 replies to this topic

[jason0429]

Hi, I was just curious that if too much ligation reaction is added into transformation would that affect the transformation??
In my experiment I get alot of colonies on my plate, but most of them does not contain my insert. I dephosphorylate my vector when i do my digestion, then i gel purified my vector and my insert. I nanodrop my vector and insert before i do my ligation. My vector concentration is normally 30ng/ul and my insert is 65ng/ul. I did a 1:2 and 1:3 ligation and O/N at 4 degree. Next day, i added 15uL ligation reaction into 50uL of my competent cells. Would this 15uL affect the up take of the right plasmids???

Another question is, in a 1:2 ratio ligation, I added 2uL of vector and 2uL of insert, is this a 1:2 ligation ratio in a 20uL ligation reaction??? and is adding 2uL of vector and 3uL insert a 1:3 ratio ligation???

Thank you

Jason

[DocFlow]

jason0429, on Nov 19 2009, 04:00 AM, said:

Hi, I was just curious that if too much ligation reaction is added into transformation would that affect the transformation??
In my experiment I get alot of colonies on my plate, but most of them does not contain my insert. I dephosphorylate my vector when i do my digestion, then i gel purified my vector and my insert. I nanodrop my vector and insert before i do my ligation. My vector concentration is normally 30ng/ul and my insert is 65ng/ul. I did a 1:2 and 1:3 ligation and O/N at 4 degree. Next day, i added 15uL ligation reaction into 50uL of my competent cells. Would this 15uL affect the up take of the right plasmids???

Another question is, in a 1:2 ratio ligation, I added 2uL of vector and 2uL of insert, is this a 1:2 ligation ratio in a 20uL ligation reaction??? and is adding 2uL of vector and 3uL insert a 1:3 ratio ligation???

Thank you

Jason

First of all, a 1:2 and 1:3 ratio is based on molar concentrations and not ng. Your insert is much smaller than your vector size. This again means that 2µl of vector is way less than 1µl insert -not 1:1. Most likely there is so much insert in your solution that the inserts ligate with each other and messes up the whole reaction. Try with less insert.

Most ligases I've used have optimal temperature at RT. Why do you ligate at 4? Also,  my favorite ligase was from Roche/finnzymes. that one used to work every time with me.

[HomeBrew]

I agree with DocFlow -- the ratio of vector to insert is based on moles, not weight.  The equation for a 1:1 ratio is:

(length of insert (in kb) / length of vector (in kb)) × ng of vector =  ng of insert needed for a 1:1 vector:insert ratio

First, you decide how many nanograms of vector you want to use in your ligation, then, if you wanted a 1:3 ratio, you would multiply your answer by 3; if you wanted a 1:2 ratio, you'd use twice as much as the answer, and so on...

[jason0429]

Hi, Thanks for replying my problem.

Okay, let say I want to use 20ng of vector, my inset size is 924bp and vector size is 5700bp and i want to do a 3:1 insert:vector, so the calculation will be (20ng x 0.924) / 5.7 x 3/1 = 9.7ng of insert is that right? Then my original insert concentration is 68ng and my original vector concentration is 28ng. So in a 20uL ligation reaction, I add in 14.3uL of vector and 2.86uL of insert. Is that right???

Thanks alot alot...

[jason0429]

is 20ng of vector good enough for ligation?? normally my original vector concentration is 28ng...