I have Isolated VIbrIo bacteriophage genomic DNA by phenol/chloroform method, I found a thick DNA band with more streak and herewith I have attached my page DNA gel picture, I need suggestion for obtaining serious outcomes.
To confirm pure phage DNA I run PCR with both the phage DNA and gDNA with primers targeting both phage sequences and non-phage sequences. Primers targeting non-phage sequences should always be negative for the phage DNA sample. Run in triplicate to avoid the possibility of a false negative.
Also, try placing less sample in the gel next time, hopefully you won't get so much streaking.
Hope this helps!
For my suggestion / guestion may i add 10 μL of DNase during the first steps of phage DNA isolation for remove bacterial /host DNA .
You definitely have bacterial gDNA contamination; may not be RNA contamination. I would suggest you add DNase and RNase before isolating phage DNA to get rid of any bacterial DNA and RNA. You can carry out thermal denaturation while monitoring it at UV260. If you get only one Tm, it is pure DNA preparation.
Best of luck.
Dear vikas jain,
Thank you for your valuable sugeston, could you send me any standerd protocol ?
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It is there in the Meanitis book. Please let me know in case you need more information. We have followed that protocol for our work. Once you have prepared your phage stock, you will have to remove any DNA and RNA that might be present along with it, followed by proteinase K digestion to remove all the proteins. Two round of Phenol:CHCl3 are then required to clean the preparation.
Vikas your DNA got smeared I think you followed a right procedure but during the time of mixing you did pipe-ting error that is why your picture got smeared
Next time when you performed this experiment make sure all all the process performed very carefully
Hi, could you please reder me to the specific method protocol you are using? (Specifically I would like to know whether you use NaI and if so, what molar concentration). Thanks,
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