I extracted my DNA (from fungi myceliAa) using 2% CTAB protocol:
- 800µl CTAB extraction buffer (2% CTAB, 0.1M Tris HCL pH8.0, 20mM EDTA pH 8.0, 1.4M NaCl, 1% PVP40, lastly b-mercaptoethanol was added to extraction buffer at 0.2% before use) were added into the samples and grind in mortar and pestle at RT.
- incubated at 65°C for 1 hours with mixing every 15m
- centrifuged at 12000x g for 10min.
- cooled to RT before adding 800µl chloroform and invert mixed (tried both chloroform and chloroform: isoamyl)
- centrifuged at 12000x g for 10min at RT.
- 600µl of aqueous layer was transferred into new tube
- add same amount of ice-cold isopropanol and put in -20°C for 15m.
- centrifuged at 12000x g 4C for 10min.
- supernatant was removed
- add 1ml 70% ethanol to DNA pellet, invert mix twice and stand at RT for 5m
- centrifuged at 12000x g RT for 5min.
- Pipette out as much ethanol as possible
- The DNA pellets were then air dried in laminar flow until DNA pellet turn partially transparent
- add in 25ul TE buffer
I ran 5ul of DNA on agarose gel with 1kb ladder. The DNA looks fine although the concentration is low. (Lane 1: ladder, lane 2& 3: DNA)
I tried amplify using ITS 1 & ITS 4; ITS 1 & ITS 2; ITS 3 & ITS 4 but nothing is amplified although the control yielded a clear band (control using control primers and lambda DNA), so it means that the PCR mix is functional.
I tried re-prepare CTAB from chemicals from other lab. I also tried increase DNA to 3 times in PCR reaction. But the result were the same, nothing amplified. I'm not sure what else to troubleshoot.
Any opinions and suggestions are much welcomed and appreciated. Thank you.
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