I isolated DNA from one of the prostate cancer cell line, its 260/280 ratio is 1.95 and 260/ 230 ratio is 1.31. After DNA isolation, I did Bisulphite treatment and after this treatment 260/280 ratio has fallen to 1.49 and 260/230 ratio came to 0.53. So, Please tell ,does the low 260/280 and 260/230 ratio of genomic DNA affect the bisulphite conversion ? and the low 260/280 & 260/230 ratio after bisulphite conversion affect methylation speecific PCR?
A high 260/230 ratio means you have rest of organic reagents in your sample, in case of DNA could be phenol/chloroform or ethanol, depending on the protocol you used for isolation, while the 260/280 ratio indicates the purity of DNA versus protein.
It seems your sample becomes poorer quality after bisulphite treatment. Bisulphite-treated DNA is so unstable and you must be really careful, keep it all the time on ice and store at -80°C. Maybe your DNA is degraded after bisulphite conversion. So every PCR, including MSP, will be affected by a low-quality DNA input, which could lead to the failure of the amplification.
Dear Lalita,
Its important to put emphasis on 2 points: 1) having a good quality DNA for bisulphite conversion and 2) Procedure for bisulphite conversion.
As Alberto explained about both ratio 260/230 and 260/280, thats imply for genomic DNA and they should be atleast 1.75-2.00 for further better experiment.
However, after bisulphite conversion, DNA become single stranded instead of double and its very unstable, so you have to be very careful with this for further experiment like MSP, sequencing etc.
Since we are using bisulphite conversion kit (from Zymo) from long time and always used proper controls with quality control, we usually don't go for nanodrop analysis after bisulphite conversion, provided input DNA should be of better quality.
I would recommend to make small aliquotes of bisulphite DNA and store them at -20 for 2-3 months and -80 for longer and don't do freezed-thaw cycle to improve your MSP quality
I store it in -20. and I put it on ice during PCR, but not getting the result. should I need to keep this DNA on ice after bisulphite treatment (After incubation at 50. ) during precipitation of this treated DNA?
After bisulfite treatment, before use, keep DNA on ice. For storage, keep it at -20°C.
The lower read after bisulfite conversion is due to ssDNA nature along with randomized degradation. Remember, the conversion requires a series of temperature treatment, enough to make the dsDNA to ssDNA. More so, the ratiometric reading is influenced by the ds nature.
Anyways, keeping the DNA constantly in cold (0'C or more) is recommended strictly. Infact, prior to setting them into the cycler, keep the PCR tubes/plate in chilled condition. This will give you crispier band. Its colloqially termed as "cold shock". All the best for your experiments.
Somnath.
Thank u for ur suggestion .I do not put PCR tubes on ice before putting them in thermocycler. will do this in next experiments. But for the ratio, ssDNA absorb more than that of dsDNA , so why is it decreasing?
I converted DNA of 115 ng/uL with a 260/ 280 ratio of 1.81. Then I got them bisulfite converted using Qiagen epitect kit. After this I got the converted DNA with a 71 ng/uL concentration and a higher 260/280 ratio i.e. 3.5. And I am planning for HRM. Please help me that what should be 260/280 ratio for successful conversion. I dont know whether a converted DNA with such a higher O.D. will work???
Hi, Neerja Aggarwal ，has you problem been solved? I use the QIAGEN Fast Bisulfite Conversion kit recently, and got the same result as yours. The 260/280 is 3.4, and there wasn't a peak around A260. I use 5 ul DNA with a concentration of 320ng/ul， and got 34ng/ul after eluted by 15 ul EB.
Hi, Ho Xianglong and Neerja Aaggarwal,
I am also using Qaigen kit for Bisulfite conversion and getting high 260/280 ratio like 2.8 and 2.7 and even for few samples 3.0.
Should I proceed these samples for MSP. I have little quantity of DNA as I am working with FFPE DNA.
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