I'm having an issue that is possibly due to the QIAamp DNA extraction?
I am extracting DNA from laser capture microdissection (LCM) of frozen section material. What I have been doing is cutting about 65 um2 and 650 um2 of material in separate tubes and then going straight into the the LCM protocol in the QIAamp Micro kit;
1) 15 uL ATL buffer applied to the sample
2) Add 10uL Prot K
3) 56 degrees overnight
4) Add 25uL ATL
5) Add 50uL buffer AL (I have been adding carrier RNA here; 1uL of 1ug/uL)
6) Add Ethanol
7-13) Washes and elute. For elution I have been warming up my water and incubating for 5 min to maximise yield. Additionally I been running the eluate back through the column again.
When I measure my DNA on a Qubit after all of this I am typically getting from 0.15 - 0.3ng/uL in an eluate of 40uL for all my samples, irrespective of the amount cut i.e. the 650 um2 samples yield roughly the same concentration of DNA as the 65 um2. Any ideas why?
Overnight incubation could be a concern.
Make sure ethanol is added to buffers.
I haven't done this before thus I have some suggestions for you.
Be sure that you have enough cells.
Do homogenization and then go on.
And over night incubation is not concern at all.
We have been using Qiagene products for DNA purification. I also found that some DNA lost after elution. Some DNA can still firmly stick to the silica-based membrane after elution. I have tried to elute them 3 times using the same steps. After the 3rd time elution, residue DNA from the membrane was still washed down. Try to minimize DNA loss, I have tried to elute them a few times and collect eluants in atube. DNA was then conenctrated with Speed-Vac to remove the extra water.
Normally, extraction using mini-kit give less NA but high purity.
SUGGESTION: 1- Having enough sample can help in recovery of the DNA. 2- For this kit, adding ethanol to the buffer may also contribute and homogenization step (ones at high speed).
Finally, try conventional method e.g. DNS if interested in getting high concentration of the DNA.
I faced the same problem when i used qiagen Dneasy blood and tissue kit for extraction of DNA from Bacteria.. DNA yield was too low and gave no results on PCR.
@ Manar Ezzat Elkhayat,
Have you checked other errors (ex. PCR conditions, primers,....) except 'low DNA amount'?? Since running PCR does not need a lot of DNA. Did you include a Positive control??
@yuan
Yes, i used another method for extraction and gave good yield with good pcr results using the same primer and the same condition.
@ Manar Ezzat Elkhayat,
Thanks for your reply. The reason I asked is because it is very hard to image that the commercial kit could not even yielded DNA amount for PCR. Qiagen products are usually considered good-quality. I checked this kit you mentioned "Qiagen Dneasy blood and tissue kit". It indeed can be used for DNA extraction from animal blood and from bacteria. Bacteria should be easy for DNA isolation. It sounded uncommon to me.
What the other method you used and succeeded? Another kind of kit?
Which tubes are you using to capture the cells? If you use ZEISS tubes with AdhesiveCap, how do you incubate overnight upside down?
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