Help me for DNA concentration for PCR - (May/30/2007 )
Dear All.
I need to set the experiment for find out the sensitivity of my PCR. I extracted my bacteria with qiagen mini prep kit, and then performed the 10-fold dilution (10-1 to 10-10) of my extraction DNA. While I also measure the DNA concentration with my extract DNA by using spectrophotomeyer before making the serial dilution.
My result was DNA A260/A280 ration = 0.86, DNA concentration = 65.3 microgram/ml. My total volume of my extracted DNA/tube was 50 microlitre. I am quite bad in calculation, so I try to calculate my DNA concentration /microlitre by using several free download programmes that were found by google. They reported that my DNA was 0.15 microgramm or 155 nanogram/mirolitre. Is it enough for performing PCR and making 10-fold diltuion, if not, Can I elute it by using elution kit for more concentration or do anything else ? please help me 
Dear All.I need to set the experiment for find out the sensitivity of my PCR. I extracted my bacteria with qiagen mini prep kit, and then performed the 10-fold dilution (10-1 to 10-10) of my extraction DNA. While I also measure the DNA concentration with my extract DNA by using spectrophotomeyer before making the serial dilution.
My result was DNA A260/A280 ration = 0.86, DNA concentration = 65.3 microgram/ml. My total volume of my extracted DNA/tube was 50 microlitre. I am quite bad in calculation, so I try to calculate my DNA concentration /microlitre by using several free download programmes that were found by google. They reported that my DNA was 0.15 microgramm or 155 nanogram/mirolitre. Is it enough for performing PCR and making 10-fold diltuion, if not, Can I elute it by using elution kit for more concentration or do anything else ? please help me
65.3 microgram per mL is the same as 65.3 ng/microlitre I don't know what the program did... This should be plenty of DNA, you can use femtogram amounts of plasmid DNA for PCR but in that case each plasmid is your target... in this case, the amount of DNA you need is dependent upon the amount of your target present/ng-ug-etc. of DNA. 65.3ng/ul*50ul=3265ng or 3.265ug DNA total isolated.
HTH good luck
Dear All.I need to set the experiment for find out the sensitivity of my PCR. I extracted my bacteria with qiagen mini prep kit, and then performed the 10-fold dilution (10-1 to 10-10) of my extraction DNA. While I also measure the DNA concentration with my extract DNA by using spectrophotomeyer before making the serial dilution.
My result was DNA A260/A280 ration = 0.86, DNA concentration = 65.3 microgram/ml. My total volume of my extracted DNA/tube was 50 microlitre. I am quite bad in calculation, so I try to calculate my DNA concentration /microlitre by using several free download programmes that were found by google. They reported that my DNA was 0.15 microgramm or 155 nanogram/mirolitre. Is it enough for performing PCR and making 10-fold diltuion, if not, Can I elute it by using elution kit for more concentration or do anything else ? please help me
65.3 microgram per mL is the same as 65.3 ng/microlitre I don't know what the program did... This should be plenty of DNA, you can use femtogram amounts of plasmid DNA for PCR but in that case each plasmid is your target... in this case, the amount of DNA you need is dependent upon the amount of your target present/ng-ug-etc. of DNA. 65.3ng/ul*50ul=3265ng or 3.265ug DNA total isolated.
HTH good luck
Thank you for your kindness. Would you mind if I ask you more questions ? I need to take my DNA for making 10-fold serial dilution (10-1 to 10-10). I need 200ul in total volume for each dilution. If I take 20 microlitre of my DNA in each dilution + 180 microlitre DW in each dilution. It is mean that my DNA concentration should also diluted in the same time as you tell me, isn't it ? My 1st (undilute DNA) should have DNA concentration = 65.3 ng/ul x 20 ul = 1306 ng or 1.306 ug DNA total , didn't it ? In my total volume in 1st dilution is 200ul, it is still have 1.306 ug DNA total or it is more 100 times diluted from this number ?
Hi,
Your starting DNA solution has, as said before, a concentration of 65,3 ng/µl.
For a tenfold dilution series you can take 20 µl of your undiluted DNA and 180 µl of water. This will give you a tenfold dilution of your stock solution with a final concentration of 6,53 ng/µl.
Take 20 µl from this tenfold dilution and 180 µl of water to get a solution with a final concentration of 0,653 ng/µl, which is a hundredfold dilution compared to your stock solution. Just go on that way (20 µl previous dilution step and 180 µl water).
But be aware that you will end up with a final volume of 180 µl for each dilution step (apart from the last one), because you take out 20 µl of each dilution for the next step. If that poses a problem you have to take greater volumes (23 µl DNA solution, 207 µl water for example). Be sure to mix the solutions after each dilution step.
Hope this helps. 
ellis
Your starting DNA solution has, as said before, a concentration of 65,3 ng/µl.
For a tenfold dilution series you can take 20 µl of your undiluted DNA and 180 µl of water. This will give you a tenfold dilution of your stock solution with a final concentration of 6,53 ng/µl.
Take 20 µl from this tenfold dilution and 180 µl of water to get a solution with a final concentration of 0,653 ng/µl, which is a hundredfold dilution compared to your stock solution. Just go on that way (20 µl previous dilution step and 180 µl water).
But be aware that you will end up with a final volume of 180 µl for each dilution step (apart from the last one), because you take out 20 µl of each dilution for the next step. If that poses a problem you have to take greater volumes (23 µl DNA solution, 207 µl water for example). Be sure to mix the solutions after each dilution step.
Hope this helps.
ellis
Ya, you're correct. Thank a lot.
