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.

Test for the correlation between boiling time and PCR

In this experiment, we test our hypothesis B: “Increasing the boiling time will improve the PCR”. (as a continuation of the last experiment)

In this experiment, Phuong’s sample is used again. (also to check her special DNA result we got last time)

In this experiment, all of our saline samples are collected with 3 spins. 200 µl of Chelex is used for all samples. All samples are filtered except Cx.

Cx is the only sample we does not filter before putting into the PCR tube.

This time, we have 4 saline samples, 2 toothpick samples, and 3 controls:

C1 – boiling at 100 Celsius for 6 minutes

C2 – boiling at 100 Celsius for 9 minutes

C3 – boiling at 100 Celsius for 12 minutes

Cx – boiling at 100 Celsius for 9 minutes  (so in theory, Cx is very similar to C2. The only difference is filter and non-filter)

TP1 – boiling at 100 Celsius for 9 minutes

TP2 – boiling at 100 Celsius for 12 minutes

+/+ homozygous control

-/- homozygous control

Here is our result:

The positive controls are quite smear because we put 20 µl into the gel instead of 10 µl as usual.

All of our saline samples work. We can actually see the correlation between the boiling times and the size of the bands. More boiling time give a bigger (and better) band on the gel:

Boiling time: C1 < C2 < C3

Size of the band: C1 < C2 < C3

The result is consistent with our hypothesis that more boiling time increase the quality of our DNA samples. More boiling time also allows more cells to lyse. Protein histone are destroyed more thoroughly.

There are not much difference between the filtered sample C2 and the non-filtered Cx. Filtering does not eliminate the smear, so I think we can conclude that the smear is not caused by Chelex or contamination. Can the smear be more likely to appear when the primers bind to the wrong site of our DNA? I wish we had time to test the annealing temperature.

The toothpick samples TP1 and TP2 in this experiment is tested at the boiling time of 9 and 12 minutes. TP1 does not give any band, while TP2 gives a very good band (the best TP result we have had this far in our experiments).

TP1 and TP2 do not give any smear.

The result from the TP samples, again, confirms our hypothesis: “TP samples need a longer boiling time compared to saline samples”.

Tomorrow, 3/10/2011, we will do one more experiment with more samples and samples from different people to confirm our result.

 

Test for the relation between the amount of Chelex and PCR

There were 3 changes we made when performed the PCR with Phuong’s samples (previous experiment):

a) Increase the amount of Chelex to match the amount of DNA

b) Increase the boiling time at 100 Celsius, from 6 minutes to 7.5 minutes

c) Increase the number of PCR cycles from 30 to 35.

Therefore, this experiment, we put our hypothesis A (increase amount of Chelex) to the test:

C1 is prepared with 5 spins (5000 µl of the saline rinse), the amount of Chelex is doubled.

C2 is prapared with 5 spins (5000 µl of the saline rinse), the normal amount of Chelex is used.

C3: 2 spins (2000 µl), double amount of Chelex.

C4: 2 spins (2000 µ), normal amount of Chelex.

C5: 1 spins (1000 µl), double amount of Chelex.

C6: 1 spins (1000 µl), normal amount of Chelex.

Cell pellet sizes

Unlike the previous experiment, the heating time at 57 Celsius is increased from 5 minutes to 8 minutes.

The boiling time at 100 Celsius is kept at 6 minutes.

Here is the result:

a) Using half the amount of primers (master mix/primer mix = 100:1)

Gel A

b) Using the usual amount of primers (master mix/primer mix = 50:1)

Gel B

In gel A, because only the amount of primer is used, none of the samples works. Even the positive control, which normally gives a very clear band, now only yields a faint band.

In Gel B, the amount of primer goes back to normal, and some of the samples begin to work: C4 C5 C6.

C4, C5, and C6 start with only a small amount of cell pellet, but it seems to work better than the big cell pellet samples.

C4 and C6 have less amount of Chelex, but they still give better band, so now we do not think the amount of Chelex really matters.

Recalling Phuong’s sample in the previous experiment, the smear we see in lane 6 probably was not caused by inefficient amount of Chelex, but caused by too much DNA template in the sample.

All samples have their heating time at 57 Celsius increased, but it does not seem to help.

Now, we think of one possibility, which is bigger cell pellet requires a longer boiling time (at 100 Celsius). More Chelex put into the sample may also reduce the efficiency of heating and boiling.

C versus TP

Looking at all the TP samples we have done so far in our experiment last night, I really wanted to figure out the difference between our saline samples and our toothpick samples. Today, I collected some cell samples using our old saline method (C) and the toothpick method (TP). After that, the samples are put under microscope for examination. Here are the taken pictures of the samples:

a) C sample:

b) TP sample:

Jonathan tried it later and got the same result. (I don’t have his pictures in my computer)

Obviously, there are differences between 2 samples. In the C  sampe, there are numerous free floating nuclei. On the other hands, the TP sample does not have any free floating nuclei. I suggest one hypothesis that our TP samples does not work because the DNA in these samples was not treated properly. Many DNA in the TP samples still stuck inside the cells (maybe the boiling process was not enough to lyse these cells). That is why the C samples, which have had more free floating nuclei already, are more likely to be ready for PCR than the TP sample.

Based on our observation in previous experiments, although many TP samples had much bigger pellet than the C samples, they did not work well (Even the HS Qubit Assay  we did early shows that TP samples had a very similar DNA concentration compared to the C samples). It proves that DNA quality plays a very important role here.Tomorrow, I will continue to check the affect of Chelex and heating to cells, nuclei, and DNA with microscope. I will also check the cells after heating at 57 Celsius  also. I will update this microscopic experiment as soon as possible.