I ran PCR using COI universal primers on DNA extracted from lice.
I added 25ul of 2x master mix, 5ul template, and 1ul each of 20uM F and R primers, with the remaining volume made up with DW to a total volume of 48ul, and ran PCR including a control group. However, no bands appeared on the gel after electrophoresis.
I then checked with a nanodrop, and all 5 PCR samples (including the control group) showed concentrations around 20000ng/ul, with A260 readings around 400, 260/230 ratios around 10-11, and 260/280 ratios around 37-47.
Where could I have gone wrong?
I would appreciate input from experienced individuals.
If you are using column purifications most use Guanidine thiocyanate as a denaturant to help bind the dna to the column. This absorbs at 260 but not at 280 so unless all of the GuT is not washed off the column you will get a small amount of dna and a large amount of Gut which absorbs at 260 . This looks like you have a lot of dna which you do not. I think the pcr is probably failing because there is too little dna. try a second wash step on your dna purification and do not overload the purification column and you shoulf get sensible readings. If this fails then you may need to clean the nano lens and make sure that the blank solution is exactly the same as the eluted dna solvent....if you are eluting with water then blank the nano with water. Similarly if eluted in TE then blank with TE. Try 25 ul pcr volumrs as small pcrs reach temperature quicker during cycling
Hi
Its possible you made a mistake in the PCR procedure, actually the times and temperatures.
you cannot make a do260 directly with the PCR; oligo and ribo nucleotides absorb at 260. if it was purified on column (but what is the point to purify if you did not see anything on gel?) then thiocyanate can be the culprit as Paul indicated.
First, I would quantify the DNA sample before starting the PCR, reduce the total volume of the reaction, and perform a temperature gradient.
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