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.

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