Hello everyone,
Currently I am working on synthesizing a single-stranded (ss) cDNA and I need to extract a specific band from gel (low melting point gel, SeaPlaque GTG agarose). I tried to follow a protocol from "Nat Protoc. 2018 Jan;13(1):195-215". There are 2 methods to extract that ss cDNA (1) using column (2) using TE-saturated Phenol. Because the affinity of ss cDNA to the column membrane is not as strong as ds DNA so I couldn't get high enough amount of ss cDNA that I need. So I am focusing on the 2nd method using "TE-saturated Phenol" as described in below
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(i) Add 200 μl of modified TE to the tube containing the gel piece and place it at −80 °C for >20 min (can be left overnight).
(ii) Thaw the sample at room temperature and transfer the solution to a new tube.
(iii) Add 200 μl of TE-saturated phenol and centrifuge (21,000g, 20 °C, 6 min).
(iv) Transfer the supernatant to a new tube, then add 20 μl of 3 M sodium acetate (pH 5.2) and 500 μl of 99.5% (vol/vol)ethanol. Vortex and centrifuge at 21,000g, 10 min, at room temperature.
(v) Decant the supernatant, add 130 μl of 70% (vol/vol) ethanol, and centrifuge at 21,000g for 2 min at 4 °C.
(vi) Completely remove the supernatant and dry the pellet. Dissolve the pellet in 11 μl of injection buffer.
PAUSE POINT Store the samples at −20 °C (short term (up to 3 months)) or −80 °C (long term (up to 2 years)).
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Currently I have some troubles in this protocol.
1/ Seems the freezing-thawing in step (i) not help to release the ss cDNA from the gel completely. I still can see a lot of EtBr signal from the ss cDNA remaining in the gel under UV light after transfer the solution to a new tube.
2/ After transfer the supernatant in step (iv) to a new tube, the clear solution turn gradually to cloudy in RT and turn faster if put on ice. I am not sure what those particles making cloudy phenomenon are, are they from the gel components? if I warm it up a little bit, it becomes clear again. That may be the reason why the author suggested to do centrifuging in RT instead of 4oC after put 99.5% (vol/vol) ethanol.
My final concentration of ss cDNA is still not as high as I wanted, just around 30-40ng/ul (I need over 100ng/ul). So base on what I observed I think I miss something in the protocol! The ss cDNA stuck in gel and the weird cloudy supernatant are suspicious.
Do you have any experience or suggestion want to share with me to improve this protocol? Anyone see the cloudy supernatant as I describe above (attached picture) when using Phenol extraction method?
Thank you very much,
Vu
When extracting with TE-Phenol should be done at higher temp (>55 deg) to let the agarose melt and release DNA in TE-Phenol (may be- The clouding you see is because of the agarose that starts gelling at lower temperatures)
You could try this protocol
http://mcb.berkeley.edu/labs/koshland/Protocols/DNA-RNA/Lowmelt.html
Hope it help.. Good luck!
Have a look on E-gel machine from Invitrogen. You will not go for gel extraction kit again, after using this procedure.good luck.
There is a big chance that the cloudy solution is due to gel, especially as it become clear when warm up. You may reuse the column several times to collect enough amount of ss cDNA.
Hi Vu,
We have always used the Qiagen QiaQuick gel extraction kit for dsDNA extraction. The Qiagen website has this information about using this for ssDNA. We have not used the QiaQuick for ssDNA, but I thought you might consider.
"As a rule of thumb, single-stranded DNA binds to silica with approximately half the affinity of a double-stranded DNA fragment of the same length under the buffer conditions used in the QIAquick and MinElute Kits. Even though no systematic experimental data exists, we expect that recovery of ssDNA fragments of approximately 200 nucleotides and below will not be very efficient after cleanup using the QIAquick PCR Purification Kit or MinElute PCR Purification Kit. By comparison, it should be possible to purify fragments longer than 140 nucleotides using the QIAquick Gel Extraction Kit.
Note that recovery of single strand DNA is influenced to some degree also by factors such as base composition and secondary structure. It has to be determined empirically by the researcher if cleanup of single-stranded DNA with QIAquick columns yields satisfactory results."
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