Basically, I want to get a segment out from genomic DNA. And put do 2nd PCR to get the product with ECorI and XhoI sites. Because the segment I want to obtain shares homology with another gene, I have to do 2 PCRs to achieve that.
I can get the single sharp band of right size from the 1st PCR. Then cut gel and purify DNA. I used this DNA as a template primer with ECorI ,Xhol sites respectively, to do 2nd PCR. I got smear band around the right size.
10X High Fidelity PCR Buffer 5 µL
10 mM dNTP mixture 1 µL
50 mM MgSO4 2 µL
Primer mix (10 pmol/ul) 1 µL
Template DNA 1 µL
Platinum® Taq High Fidelity 0.2 µL
Water to 50 µL
PCR condition as follow:
94ºC 2 minutes
30 cycles of:
94ºC for 30 seconds
55°C for 30 seconds
68ºC for 2:30minutes (2590pb)
4ºC hold.
Can somebody give me some suggestions?
It could be the annealing temperature try to raise it up according to your primer TM.
Also check the purification kit and try to dilute the first pcr product 1:5 and use 1 ul from it directly without gel purification as long as there is no non specific bands appear.
Hi Diogo. I loaded 40ul on the gel. The concentration of 1st PCR product is 22ng/ul after purification. I tried 1ul ,2ul and 4ul to the mix. But the same smear. I will try another cycle next time. .Thanks a lot.
Hi Yasser. I did couple different annealing temperature according to the primer Tm. But I could not get the band I want it to be.
Used it directly from the PCR tube without purification? But I think it is not that good. Right?
Thanks.
Hello Haoran. I think the amount of 2nd. PCR template was too much and you had better decrease 2nd. PCR cycles. Good luck.
When you run a gel with smaller fragments, you need a harder gel. Consider increasing your % agarose, and use a lower voltage. Small DNA frag can diffuse easily out of the gel matrix.
Hi Haoran. gel purification may affect the pcr reaction you can purify the pcr reaction without gel purification. There are a lot of kits that allow you to purify the pcr reaction directly.
Hi there,
The first thing I would do (quicker and easier) is to increase the annealing temp. Your are currently using 55°C for 30 seconds,
I would go for 58°C for 30 seconds, and if you notice that it is getting better I would even go for 60°C for 30 seconds.
Good luck!
you should adjust the concentration of your DNA samples:
1-the concentration which you add to the PCR Mixture.
2-How much of the DNA you use to run the gel.
Thanks guys. It is a problem with the primer. I changed the primer. It is ok now. :) Thanks.
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