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.

 

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