DNA - Ball Python DNA Amplification with TFEC primers

REAGENTS

• 10X PCR Buffer
• MgSO420millimolar (mM)
• Taq Polymerase 5U/ul
• Primer Tfec exon5 Left 10micromolar (µM)
• Primer Tfec exon5 Right 10micromolar (µM)
• DNTP's 10micromolar (µM)
• Ultra Pure Water
• 100bp ladder (100-2,000 bp)
• SYBR Safe DNA stain
• Loading dye
• Agarose

EQUIPMENT

• DNA LoBind tubes 1.5mL
• PCR 8-Strip tubes 0.2mL
• Micropipettes
• Thermal Cycler

MASTER MIX

Below is the recommended volume for one sample. Adjust for the number of samples to process and add 10% to account for pipetting error. Thaw all reagents at room temperature before using them except the Taq DNA polymerase, which has to be kept on ice at all times. Premix all reagents and add the Taq DNA polymerase at the end, vortex thoroughly for 0h 0m 30s seconds. Aliquot the master mix in to each tube 28µL and then add 2µL of DNA sample. The final volume for each reaction is 30µL .

A	B
Reagent	Volume (uL)
10x PCR Buffer	3
MgSO4	4.5
Taq Polymerase	0.3
Primer F	1.5
Primer R	1.5
DNTP's	0.6
Template-DNA	2
Water	16.6

Table 1. Master Mix for PCR.

PCR CONDITIONS

A	B	C	D
STEP	TIME	TEMPERATURE (ºC)
Initial Denaturation	3 min	94
Denaturation	15 sec	94	25 Cycles
Annealing	30 sec	53.2
Extension	30 sec	74
Final Extension	2 min	74

Table 2. Thermocycler program

AMPLICON QUALITY CHECK

1. Prepare a 1% agarose gel with 1X SYBR Safe DNA stain.

2. Add 4µL of 100bp ladder (100-2,000 bp) in the first well.

3. Premix 4µL of sample and 1µL of 10X loading dye or 3µL of sample and 1µL of 6X loading dye. Load this mixture in each well.

4. Run the samples for 0h 35m 0s at a 100V.

5. Visualize in a trans illuminator and take a photo of the gel.

6. The product should be a clear, single band around 200 bp.