I sorted human PBMC, then washed the cells and stored at -80C as dry cell pellet last year.
This year, I pulled out the dry cell pellet from freezer and lysed with in-house lysis buffer. The recipe is 10 mM Tris-HCl pH8.0 with 0.5% v/v IGEPAL (NP40), 0.5% v/v of Tween 20 and 0.5 mg/ml Proteinase K (working concentration). I lysed the cells at ratio of 10,000 cells per ul lysis buffer, at 55 C for 2 hours, followed by 85 C for 15 min to inactivate Proteinase K.
This is a standard protocol in our lab that I've been using. I have old batch of cell lysate that worked nicely. This time the lysis buffer was freshly made. The surfactants were taken from the same stock bottle. For 10 ml of 10 mM Tris, I added 50 ul of IGEPAL (NP40) and 50 ul of Tween 20. Proteinase K was RNA grade, same batch, expired in late 2018 but had been kept at -20C and was fine in 2019.
After lysis, the lysate was not further purified and proceeded to PCR. Usually I do other complicated PCR but since I had problem amplifying my target, I checked its b-actin. I found that the b-actin Ct came up very late in the new batch of lysate. My lysate all have around 10,000 cells input per ul. Their b-actin usually came up at 17 cycles, which makes sense compared to the plasmid standards. However, this time, the b-actin came up >27 cycles and some even 32 cycles. This is like a 1000-100000 times less than expected, as if there is no DNA in the lysate.
I am pretty sure there were cells in the tubes. I did purity check after cell sorting and I can see the cell pellet before I added the lysis buffer.
I have done a serial of troubleshooting to rule out the possibility of PCR being inhibited by components in the cell lysate, including spike in the cell lysate in positive control, diluting the cell lysate, etc. PCR reagents are all fine as I have controls in the same runs.
I also take aliquots to further treat the lysate in case it was not lysed properly the first time. I set up 7 conditions:
Untreated
Heat at 55 C for another hour with inverting every 15 min.
Heat at 55 C for another hour with vortex every 15 min.
Add more Proteinase K to lysate, heat at 55 C for another hour with inverting every 15 min.
Add more Proteinase K to lysate, heat at 55 C for another hour with vortex every 15 min.
Add more Proteinase K and lysis buffer to lysate, heat at 55 C for another hour with inverting every 15 min.
Add more Proteinase K and lysis buffer to lysate, heat at 55 C for another hour with vortex every 15 min.
These conditions were to check if physical force like inverting or vortex help to lyse, and if more surfactant and or Proteinase K help to lyse. Unfortunately none of the above approaches worked. The b-actin came up even later after the treatment.
Now I've run out of ideas of why there seemed to be very low DNA in my cell lysate, especially that this protocol has been used and worked. I also don't know what to do with my current batch of cell lysate. Is there any way I can save these sample? These are precious samples and all the cells were gone.
Since I did not do further purification with the cell lysate, everything should still be in the tubes, whether or not the cells were lysed completely. I even thought of an extreme scenario that the samples were degraded by bleach for some strange reason... but spiking in the positive control did not affect the PCR at all almost rule out this possibility.
Does anyone have any idea of why that happen and how to salvage the samples? Much appreciated!!!
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