ligation and proof reading Taq - (Oct/07/2006 )

I am doing a reporter assay for a variant in a promoter region and so I decided to use a proof reading taq to amplify my 1.5kb region of interest to be used for ligation and cloning into a reporter vector. When selecting the colonies by ampicillin resistance and digesting them to confirm that the insert was ligated to plasmid it seems that ligation efficiency was very low (from 70 colonies selected I only found 11 that took up the insert). I am thinking to increase enzyme concentration when generating the sticky ends of plasmid and construct as I think that this might be due to incomplete digestion of either one of them or both. Any suggestion please?
Also when sequencing the plasmids from colonies that took up the insert I found a number of random errors even though I used a proof reading taq.
does anyone encountered such a problem with proof reading taq? I used one from SIGMA so it was not a cheap one :-(
any suggestions will be greatly appreciated

Thanks in advance

I use KOD hifi.... it is a "relatively cheap" proofreading DNA polymerase. I have yet to get any error from this polymerase for PCR products 3kb and below. Proof reading Taq... that just sounds weird....

As for efficiency, well just make sure the DNA is properly cut, the vector must be gel purified to remove denatured plasmid (which are resistent to restriction enzymes) and all the DNA fragments kept cool and clean to prevent nuclease and thermal degredation.

Finally, improved ligation efficiency can be made by increasing the insert:vector molar ratio.

But still, I wouldn't be too worried about 1 in 7 efficiency.... unless this is via a miniprep screen. And if you are doing a miniprep screen, consider doing a colony PCR screen. It is much faster, takes a whole lot less effort, and a 96 colony screen takes as much effort as doing a 24 colony screen.

thanks for information. regarding the first step I am extracting digested linear plasmid and products from gel, which are then used for ligation.
following transformation I am selecting colonies, grow them in broth then doing a pure culture plate from which I select a single colony. i inoculate another broth from which I prepare a miniprep. I check the miniprep for presence of insert by double digestion and the I check the insert by sequencing.
A very long procedure, what about colony PCR?

thanks.

Here is a brief discription of my colony PCR protocol

- Ligated DNA is transformed into cells

- Plate transformed cells on selection plate

-go home. wait 13hrs

- Pick a well separated single colony from selection plate via yellow pipette tips. Move said colony to 96 well microtittle plate. Place individual colony into a single well containing 50ul sterile distilled water. Repeat for as many colonies as desired.

-make up PCR mix. Use multichannel pipette to add 7.5ul PCR mix to a 96 well PCR plate. Add 2.5ul colony solution

- run PCR reaction. (Do something else while waiting)

-add 4ul loading dye once PCR run is complete. Load (PCR reaction mix + dye) into gel and run.

And that is about it. Complete run takes about 5 hrs... most of which does not require your direct attention.

As for the current protocol you're following, the pure culture plate step and the broth step associated with it can be dropped. It is an extra step that is not required. As long as your colonies are well separated there isn't a need for the pure culture plate. Use more plates in the intial transformation selection step. I use no less then 2 plates (85mm radius) usually 3, sometimes 4. And if I need good separation for all my colonies I use a 22cm X 22cm plate.

thanks for the information.