Hi,
I am trying to do a PCR from transfected HEK cell containing my protein of interest, however I am not getting any success, while the control DNA is giving the correct band with same primers and cycling condition, but not the sample I am using directly from HEK cell.
Therefore, I would like to know if anyone have any experience in performing PCR with directly from HEK cell lysis without DNA isolation.
Thank you.
Manisha Shahu Please take a look at this protocol shared by Jeroen Strating.
* HeLa cells, 1/6th of a confluent well in 96-well format.
* Trypsinise cells & take aliquot in small volume (e.g. 50ul). Pellet cells (1min full speed in a table top should suffice) and remove supernatant.
* Wash by resuspending in 5-10mM Tris pH8.5, pellet again.
* Resuspend cells in 10ul of the above Tris buffer, heat 15' at 99 degrees Celcius.
* Cool on ice, centrifuge 1min at full speed.
* Use the entire supernatant directly in a PCR.
- We have tried a PCR (reaction of 50ul total) with Q5 polymerase (NEB), but I suppose that e.g. Taq should also work. Conditions may differ for each PCR product, therefore first optimise your reaction on purified DNA and don't forget to take along controls: purified DNA as a positive control and water-only as negative control.
- We didn't try less input material, but bands are very fat. So, you could probably scale down even further.
- Personally, I would not useTE, as EDTA might inhibit subsequent enzymatic (PCR) reactions.
- As mentioned above, ProtK may be needed if you use (much) more material.
This method has worked for me too.
You can go to the original thread - https://www.researchgate.net/post/Can_anyone_share_a_good_protocol_for_direct_PCR_of_genomic_DNA_from_mammalian_cells_without_DNA_extraction
Akhil Baby Thank you for the reply and suggestion. I tried doing PCR using almost same approach, it's just I boiled my sample in water instead of Tris buffer and also tried with Q5 and Taq both enzyme, but unfortunately it did not work.
Manisha Shahu Have you verified with a positive control for your PCR reaction using purified DNA as a template?
Akhil Baby Yes, I am getting very nice PCR band with purified DNA as control.
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