Help-loosing DNA after the cleaning - (Oct/29/2005 )
Well, if you feel you must continue to use two enzymes that each generate ends compatible with the other, then you're going to have to proceed the way you have been.
I honestly can't imagine why you'd want to do that -- look at all the extra effort and time you've expended chasing what should be -- given the proper enzyme choices and PCR primer design -- a routine cloning.
But, it's possible I'm missing some piece of data particular to your experiment. Good luck!
I honestly can't imagine why you'd want to do that -- look at all the extra effort and time you've expended chasing what should be -- given the proper enzyme choices and PCR primer design -- a routine cloning.
But, it's possible I'm missing some piece of data particular to your experiment. Good luck!
I really agree with Homebrew here. Although I do not possess all the data relevant to your experiment, it seems like it would be worth it to shell out another $60 for another restriction enzyme that does not create this problem.
How much do you value your time? I know that I value my time at more than $12/hr. I think it will take you more than five hours to resolve this problem using the current technique as opposed to the alternative.
Opportunity cost is a very important consideration in conducting molecular biology. Sometimes it is very much worth it to spend the extra money to save a few weeks of your valuable time. Shop around and save on the things you can save on, but do NOT scrimp on the essentials (like the appropriate restriction enzymes for the task at hand).
-Matt
Hi to all, thank you for your answering.
Finally I did the ligation of the new-created chimera with a plasmid and subsequent transformation to DH5a, with the ratio 1:3 and 3:1, and seed to a plates with resistance, with two negative controls-
1. No DNA
2. Plasmid cutted without insert..
Neg. controls worked OK-no colonies at all and 20-30 colonies in the plates with inserts grown.
I picked 6 colonies from every plate for PCR screening, and in PCR program the first stage 94 degrees I did for 10 min to destroy the bacterial membrane.
The primers I took were the primers from both sides: sense of A and anti sense of B. But in the PCR I get Nothing.
What do you think whether the negative PCR is a sufficient answer –it means there is no chimera and I have to go back or there is another way to check ? Thank you.
I gather you are doing colony PCR on your picked colonies. Almost always people who are new to doing this think that they should use lots of cells. I pick a small amount of a colony into 100 ul of DI water, vortex it, and use 0.5 ul of this solution as a template for a 10 ul PCR reaction. Plate out some of the water solution onto a master petri dish plate. Too many cells can inhibit the PCR reaction.
Unless I were certain my colony PCR was working, I would just prep DNA from several colonies and run them on a gel, or cut and run them on a gel. Quick and dirty preps can be used for this.