Hello all.
Around two years ago I extracted DNA using a qiagen kit from a DT40 cell line culture. Yield was around 100 ng/ul with a 260/280 ratio around 2. I then used the epitect kit to treat the DNA with bisufite. I used that sample for PCR for 4 different genes (amplicon sizes 312, 324, 551 and 661) and I succeeded in getting the amplicons I expected. The cleaning and subsequent sequencing procedure also went well. Meaning I had the product after cleaning and using either the forward or reverse primers for sequencing I was able to get a good sequence with a low background. Recently I have been trying to use the same protocol for DNA extracted using TrizolLS from a primary chicken B cell culture. Yield was around 200 ng/ul with the 260/280 ratio around 1.5. The sequencing results were problematic. The two shortest amplicons were almost completely sequenced. However, the two larger ones were not. Having primer dimers or a high background. When I used the nanodrop (set to RNA) on the DT40 converted samples I got between 5 and 10 ng/ul. most of the reads on the new samples were either below 2 or negative. My Questions are:
1. In the collective experience, is kit DNA extraction usually better than TrizolLS extraction in terms of quality?
2. Can that difference in quality carry over to a poor bis converted yield, or higher fragmentation of the DNA in the process? would that explain not getting some of the longer amplicons?
3. How much of a factor are the lengths of the amplicon I am after? I saw some articles discussing 300 as the limit and some discussing 500 bp as the limit for bis converted PCR amplicons. However, I was able to get those amplicons from the DT40 samples.
Thank you all
Based on your 260/280 ratio you have residual ethanol in your DNA. That will interfere with bisulfite and with downstream applications. Usually this is more a problem with manual DNA extraction than with a kit. That being said if you dry your pellets longer you may get a better result. I usually use kits for any sample I plan to bisulfite.
If your DNA has ethanol contamination then yes that is carrying over to a poor bisulfite conversion yield. If I were you before moving on to sequencing I would be running a PCR for a housekeeping gene maybe B actin as a measure for bisulfite conversion quality.
It is likely that the poor bisulfite yield is causing the issue with the sequencing but you are a little ambitious seeking larger amplicons. Reducing the amplicon length from 600 to 300 and you may have less problems in general even with a less than stellar yield of bisulfite converted DNA. As you have gotten them to work before I'm guessing you were either really really really lucky the first time through or the DNA quality and bisulfite yield was much better to begin with. As sequencing can go wrong at so many steps I'd say focus on getting high quality DNA and bisulfite conversion first before worrying about amplicon length. GOOD LUCK!!
- Badges
- Science method