Hi,
I need to extract viral RNA from swab samples using solid phase extraction (SPE) method. In order to optimize the microfluidic chip and reagents, I use short DNA sequence (~100 bp) to test the binding affinity of the SiO2 surface. The SiO2 surface was cleaned using IPA and water before experiment. Then I measure the DNA concentration before and after SPE. Here are the steps: 100 uL of sample+chaotropic agent(1M guanidine thiocyanate) -> 100uL of 70% Ethanol-> pull air through the channel until the channel is dry-> 50uL of nuclease free water. I thought ethanol dissolves guanidine thiocyanate and will evaporate fast so there shouldn't be any liquid left. However, I got about 40% DNA recovery but always have a lot of contaminants (the 260/230 ratio is very low), which affect the PCR downstream. Does anyone have any idea how to remove the impurity?
Thank you!
Hello, Yi-Hsuan.
You have to describe reagents procedure in detail so that people could provide a real solving advice.
For example, what are the chaotropic agent? And how many cells, what volume of each agent!!! How did you determine the efficiency of DNA recovery? Have you absolutely cleaned the surfaces of solid phase? If the inner surfaces of the device have a small amount of remaining chemicals that was used during fabrication. You may be using interferring organic or inorganic agents, maybe gases, too. It should be EXTREMELY cleaned and dried completely. The inner surfaces where the biological cells or biomolecules would contact. All these matters give some clues to RG colleagues what may have happened.
Hi Dr. Lee,
Thank you so much for your valuable advice! I've modified my question and hopefully it is more clear.
Since SPE works for both DNA and RNA, I use a DNA sample (~100bp) with known concentration for testing and use nanodrop to measure the concentration before and after introducing the sample to the chip.
Hello, Yi-Hsuan.
I understand that your concerns are i) low DNA recovery and ii) possible contamination in the finally pulled solution. You can solve the problems by correcting or modifying procedures step wise in your hands. You've done what required. Just check each step to see what might be wrong.
i) Give enough time to allow DNA settles by checking each collected drain of GuSCN, 70% EtOH and H2O (increase vol to 100uL to get maximum DNA) through taking time to pull them and dry for next use. Proceed DNA extraction in 3 separate Eppen-tubes of each drain by conventional macroscopic method. Measure DNA by Nanodrop. Then you know what step you should have given more attention, probably give more time, or give more washes like 70% EtOH(I) and (II) washes, 100uL each. The results will tell everything and instruct what you have to do. [I would like to give more time for GuSCN sol (probably much longer for real viruses) to guarantee firm binding to solid surfaces, one more 100 uL 70% EtOH wash for full recovery, and 100 uL H2O free of nuclease, collecting all drainage solutions for EtOH precipitation of remaining DNA].
ii) You may get a funny reading on Nanodrop when you have microscopic strings, particles and dusts in your collected DNA sol. All the interrupting culprits are derived from the tubes, microfluidic device prepared. When you wash, the culprits are still there due to their non-specific electrostatic properties to the surfaces of tube walls, devices, pipette tips, in the H2O you are preparing for solutions and even in the commercial products of Millipore filter. My suggestion is to use an air blower before you use any containers. Just blow away!!! Because they survive even in spinning down at 12,000 rpm in the bottom of the tube.
Have a look at the link. https://www.researchgate.net/post/Any_idea_what_is_clogging_my_needles_used_to_inject_Morpholino_into_zebrafish_embryos
Best wishes
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