hello
I want to extract DNA from human serum and blood. unless I tested different extraction methods (such as phenol/chloroform; glass beads; kits; direct PCR on serum samples after some treatments) but I could not get any band in PCR. The results of nanodrop examination showed good results but the PCR results were negative. It should be say that in other examination I get PCR bands which had very weak. Could any one help and guide me?
best regards
Almost all of the dna that you purify will be degraded human dna and very little will be bacterial. You might want to run nested or hemi nested pcr on these sample to get round the low template dna problem
Nanodrop is very sensitive machine... And its data is not reliable for measurements of DNA as at 260nm many compound show absorbance. Check 260/280 ratio. For PCR,
Take positive control for same bacterial DNA and negative control of human DNA. This will rule out your primers are working fine or not. Than if small or very less intense band apear than control will tell it polymerizing human gene or bacterial.
Nanodrop will give you only concentration only, but of the integrity of DNA id tempered, it will not tell you. to check DNA integrity, you need to run hi
Nanodrop will give you only concentration only, but of the integrity of DNA id tempered, it will not tell you. to check DNA integrity, you need to run h
This is in case if you were able to get the DNA extracted from bacteria.
Another point, i am concern is, If you were not able to extract bacterial DNA i.e. the intact DNA in your hand may be the host DNA. So, there would be no amplification of bacterial primers.
If you get intact DNA by gel and yet not getting bacterial primers amplification by PCR, try to run some host gene primers on the DNA and see, if its host DNA it will give you amplification. By this way you will be able to rule out DNA extraction from bacterial cell.
If you get nothing from human gene primer even after having intact DNA, than the concentration of bacterial DNA is very low, to solve this issue you need to run Nested PCR as suggested by Paul Rutland .
WIsh you all the best.
hello
thanks a lot for all your responses.
1. I used positive control from culture medium and the PCR results were good but (i.e. my primers are ok.) in clinical samples i could not extract bacterial DNA with the same method as control positive.
2. I sent my samples to another lab and they extract its DNA and i get PCR bond but they don't tell the method to replicate.
3. I run it on agarose gel but did not see any bond....
best regards
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