PCR of ligation products - (Nov/30/2005 )
I am having a problem amplifying specific ligation products with PCR. The technique is as follows:
-amplify gene from genomic dna
-digest amplified product with two different restriction enzymes and gel purify the left and right gene borders (ca. 1kb each), each having one blunt and one (different) sticky end
-digest a plasmid using the above two restriction enzymes to free a fungicide resistance cassette
-ligate the cassette together with the left and right genomic dna borders
-PCR amplify the ligation product using the same primers as in step 1
The amplified product is then used for fungal transformation by homologous recombination. The problem is, after the final pcr step all I get is small non-specific bands. The correct band would be about 6kb, so I'm using a long-range pcr kit for the last step. Does anyone have suggestions on how to overcome this? I hope to graduate someday!
I wonder if you have checked the successfulness of your ligation. If the ligation didn't work in the first place, you may not be able to get PCR product. You can run a gel with the ligation reaction after ligation.
I only did a 10 muml ligation, do you think I should run the whole thing and then purify the correct band, or scale it up and run an aliquot?
Just run part of it, you will be able to see something on the gel. Remember to run controls (such as without ligase) alongside with the ligation reaction.
Cheers, I'll tell you if it works.
After ligation, the recombinants become non-linear DNA, how could we determine if the ligation is successful as the ladder only tells the size of linear DNA?
The product should be linear, I'm not cloning into a plasmid.
I got it to work by running the ligation on LMP agarose and using a gel stab as the template for PCR. Thanks for your help!
It sounds like you got your construct. Congrats. I've made a lot of KO constructs for fungi as well. We would do some high through put KOs and found a Three Part PCR strategy was very easy and in some cases more efficient. Also called overlapping PCR (among other names).
If I recall correctly, what we did was PCR the arms (genomic flanking regions) with long oligos that have overlapping sequence to our marker and purified. We extractedthe fungal marker (resistence gene, auxotrophic marker, whatever) from a plasmid. We added all 3 fragment to one PCR reaction in an optimum molar ratio to drive the ligation and PCR amplification of the full length KO construct.
We used a XL type Pfu to ensure accuracy and robust amplification of constructs usully about 3-4 Kb.