Bisulfite PCR - Testing Primer Annealing Gradient with Lake Trout Bisulfite-treated DNA

INTRO

Important

This PCR was done incorrectly! The cycling program was setup incorrectly. Notebook post is being left for posterity.

Well, nothing like starting off a notebook post with a callout indicating this was done incorrectly… I’ll leave all of this here anyway.

After previously designing bisulfite PCR primers for the C1Q gene on 20240709, it was time to actually test them out. Due to their length, low complexity, and low melting temperatures (T_m) I opted to test these out on a pool of the Salvelinus namaycush gDNA isolated on 20240712, using a temperature gradient.

MATERIALS & METHODS

DNA Pooling

1uL of each sample was used to create a pool of gDNA.

PCR Reactions

PCR reactions were run using EpiMark Hot Start Taq DNA Polymerase (New England Biolabs), according to the manufacturer’s protocol, in a total reaction volume of 25uL, using 1uL of the pooled gDNA.

Since I was testing three different annealing temps (45^oC, 50^oC, and 55^oC), I ran reactions in duplicate for each temperature (i.e. a total of six reactions).

PCR Cycling

Annealing temperature gradient PCR was performed in a CFX Connect (BioRad).

Cycling params:

Warning

The cycling program is incorrect and never actually goes through cycling!!!

Figure 1: Screenshot of cycling program, also sdhowing temperature gradient.

1. 95^oC - 30s
2. 95^oC - 15s
3. 45 - 55^oC - 20s
4. 68^oC - 4m
5. 68^oC - 5m
6. 4^oC - FOREVER
7. Go to Step 2.

Samples were loaded in the following positions:

Figure 2: PCR tube placement in thermacylcer

PCR reactions were loaded on a low-TAE, 1.0% agarose gel, with ethidium bromide. Gel was run at 107V for ~45mins and then imaged.

O’GeneRuler (ThermoFisher) Ladder was used (5uL) for size reference:

Figure 3: Ladder

RESULTS

No amplification.

Figure 4: Agarose gel with pairs of lanes marked with PCR annealing temperatures of 45^oC, 50^oC, and 55^oC

Figure 3: Ladder