Cheek cell incubation time
Does the incubation time at 100° C affect the success rate of PCR?
Based on recent observations under the microscope, it may be possible that the reason why we have been unsuccessful with getting a consistent product may have something to do with the duration the cells are incubated at 100° C. We stained some cells with methylene blue before and after incubation to see if the incubation time affected the cells.
Fig. 1. Cheek cells before boiling (20x Mag)
Toothpick sample, Prior to incubation
We have observed that prior to incubation toothpick samples contain primarily intact cells. (Fig 1).
Fig. 2. Toothpick sample, incubated @ 100° C (9 min)
Toothpick; Incubation at 100° C for 9 minutes
Cells collected by toothpick become transparent after incubating for 9 minutes (Fig. 2), suggesting that the cells have been lysed.
From the first two pictures (Fig. 1 and Fig 2), you can see clearly that the cells collected by toothpick method stain differently with Methylene Blue after being incubated with chelex at 100° C for 9 minutes.
Fig. 3. Saline Rinse (100 degrees for 9 min)
Saline Rinse, 100° C for 9 minutes
In the image of cells from 0.9% saline rinse (Fig. 3), you can still see intact cells with a nucleus still visible inside.
Fig. 4 TP sample. Most cells empty.
Toothpick, 100° C for 12 minutes
Many of the cells incubated for 12 minutes were lysed (Fig 4).
Fig. 5 TP Sample. Cells empty.
Toothpick, 100° C for 12 minutes
Similar to fig. 4. Many of the cells incubated for 12 minutes were lysed (Fig 5).
Does the incubation time at 100° C affect the success rate of PCR?
Longer boiling may be necessary for complete cell lysis, and the boiling time needed for saline rinse-collected samples may be longer than that needed for toothpick-collected samples.
Brian McCauley
josemarte17
Minh Nguyen
akomirenko
xlinda07
Alex Chao
jonathanmai
Phong Lu
tszcchow
Phuong Vu
I edited Jonathan’s post for clarity. I added the concluding sentence: the boiling time needed for saline rinse-collected samples may be longer than that needed for toothpick-collected samples. Is that true? It seems to fit these pictures, but it doesn’t fit with the PCR results.
Perhaps the reason why the conclusion doesn’t fit with the PCR results is that we forgot to take into account the free floating nuclei in the saline rinse samples prior to incubation.
The PCR results suggest that collecting cheek cells by saline rinse give better results than collecting cells by toothpick.
The free floating nuclei are also membrane bound which may explain why we can still see them under the microscope and as soon as we incubate the sample, the DNA contained within the nuclei would dissolve into the solution. Which explains why we seem to get PCR with saline rinse and not toothpick.
For any sample still containing intact (unlysed) cheek cells, we have discovered that at least 12+ minutes of incubation at 100C is necessary for adequate lysis of the cells and release of DNA into solution.
I think we should not depend on these pictures. I think the boiling time for each sample depends on the size of the pellet and the amount of Chelex we add into the tube. Recalling Leo’s rerun PCR, the samples with 1 spin worked. Samples with 2 spins gave faint band, while the 5 spins failed. We also noted in that experiment that samples with 2x amount of Chelex were worse than 1x. We only boiled 6 minutes for all samples. 6 minutes maybe enough for small size of pellet, while bigger cell pellet and/or more Chelex requires longer boiling time.
We boiled 7.5 minutes for Phuong’s samples (the first one gave us good result). Those samples only had a decent amount of pellet, not much.
We should also note that collecting cells with toothpick always (and easily) gets more cell (bigger pellet) compared to saline rinse method, which requires 4 to 5 spins to get the same amount.
On the contrary, I think the pictures do help give us inportant information about our samples. We know that methylene blue is used as a safe way to stain cheek cells and reveal the nuclei (source: http://serc.carleton.edu/microbelife/research_methods/microscopy/cellstain.html) Methylene blue also has an alternative function of staining proteins and enzymes within a cell if they have been denatured. For instance, in the cheek cell samples that have been boiled for 12 minutes, we can see that they have a very vivid blue stain but no visible nucleus indicating perhaps the boiling was sufficient to denature proteins and enzymes most importantly the DNases that could potentially degrade our DNA samples.