Dear fellow scientists,
I like to performed a direct amplification of a fragment of 222-bp DNA from human genome and use is for further analysis. By using the following methods, I obtained amplicons of the target size for a few times, and then I repeated the same experiment with the same procedure, but I only got smears. I tried to use fresh primers, fresh polymerase, and freshly prepared samples, but nothing helps. Since then I only get smears. It would be really amazing if anyone knows what might be the reason.
The method I tried was:
Component (total volume: 25 μl)
- 10 µM Forward Primer: 0.5 µl
- 10 µM Reverse Primer: 0.5 µl
- crude cell lysate: 5 µL
- OneTaq 2X Master Mix with Standard Buffer: 12.5 μl
- Nuclease-free water: 6.5 µl
PCR program (touchdown PCR) is described in the attached picture.
Hi,
Double check everything (exactly everything) for a DNAse contamination. Water, buffers, pcr reagents etc.
Good luck
I think that you have some post pcr contamination of previously amplified template in your reaction. The amplification of this contamination is very efficient and the over amplification is causing the product to self anneal and form longer chains of amplimer. The contamination could be in a reagent or in your pipettes pr in the dust or plasticware in your working area. To test this run another pcr exactly as you have already done but include 3 tubes where you amplify water instead of dna sample. If you get an amplified band when there is no template dna then you do have contamination. Every 10 cycles of pcr is 1000 times more product so even invisibly small amounts of contamination completely spoil a pcr result
This is just the case of "TOO HIGH" DNA. Try to reduce the DNA concentration and adjust it to 20-30 ng / 25 µl reaction. If you have a nanodrop, try to take a rough reading of the DNA concentration of your cell lysate.
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