SYBR™ Green PCR Master Mix

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Amplicon size should be consistent for each target sequence and limited to approximately 65 - 100 bp.	PCR plates, tubes and pipette tips should be UV sterilized for 20-30 mins

Upstream tips
Amplicon size should be consistent for each target sequence and limited to approximately 65 - 100 bp.
Protocol tips
PCR plates, tubes and pipette tips should be UV sterilized for 20-30 mins

Publication protocol

RT-PCR
RNA was extracted using Trizol (Invitrogen) according to the manufacturer’s instructions. RNA (5μg) was used for cDNA synthesis utilizing the Superscript III strand synthesis system (Invitrogen). For qualitative analysis of POLA1 transcripts, the following primer sequences were utilized: primer A (exon 10, 5’-AAAGGGGCAGATGAGGAACAA-3’); primer X (exon 13a, 5’-TCTGACAGTGGTGATGAAAAG-3’); primer B (exon 15, 5’-ACAAG CGGTGGTGGACTGAC-3’). Quantitative real-time RT-PCR was performed using SYBR Green based detection (Invitrogen) and a Mastercycler (Eppendorf, Germany) as previously reported46. Experiments were performed using technical duplicates or triplicates, data were normalized to housekeeping genes and the relative abundance of transcripts was calculated by the comparative ΔΔCt method. All primers used for qRT-PCR are indicated in Supplementary Table 6.



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Manufacturer protocol

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