I am planning to obtain human blood samples in EDTA tubes which will be followed by column-based DNA extraction (DNEasy extraction kit). These samples will be subjected to FlaB nested PCR to check for pathogenic leptospirosis DNA. However, the transport component of the blood tubes is problematic. There is a risk of the blood samples being transported/left in room temperature for some hours. Based on your experience,
1. how long can I allow the samples to be left at room temperature, in order to get accurate results?
>> Room temperature should not effect quality or quantity or DNA.
2. Does DNA quantity decrease with time at room temperature?
>> Quanity - NO, quality - can be if left for longer durations like weeks or months
3. Do the PCR results after gel electrophoresis get affected (Ex: false negative for FlaB PCR) due to this?
>> False positives and other hits depends on primers and not on DNA quality primarily
4. Also, is it advisable to extract DNA from samples that are kept at 4C for upto 48 hours? Will this also affect the PCR result?
>> Above answer have already resolved this question I suppose
If you are using a DNEasy extraction kit, the quantity and quality might be affected with hemolyzed samples. A keen treatment before transferring the samples to the columns is recommended not to cause blockage of the spin column filter paper pores and further reduce the quality of DNA.
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