We have found that SGI inhibits amplification of medium-size genomic DNA fragments and that this inhibitory effect can be reduced by using a PCR mix, denoted here as mix IV, with modified salt composition [ 5].
Reverse transcriptase (RT -PCRs foRT -PCRs expression analysis oforSXL3 were callele out using a PCR mix (BIO-X-ACT Long Mix; BiolinexpressionLtd, London, UK) analysisers (ASXL3_RTmut_fof 5'-ASXL3CAGTTGCGGATAAGG-3' and ASXL3_RTmut_rev 5'-TGGGGTTCTTCATGAGAATTC-3'), located in exons 10 and 11.
PCR amplifications were carried out in 10 µl PCR reactions using a PCR master mix (QIAGEN, Inc ., 4 µg Bovine Serum Albumin (Sigma) and 2 µM primers using the following program: Initiation at 94°C for 10 min, followed by 94°C for 30 s, 49 60°C (annealing, depending on the primer pair, see Table 1) for 45 s, 72°C for 50 s, followed by 10 min at 72°C, repeated for 59 cycles.
A 959-bp internal fragment of lacIIC was amplified using a PCR master mix (Thermo Scientific, St. Leon-Rot, Germany), chromosomal DNA of CJ18 as template and the primers lacIIC_for and lacIIC_rev (Table 4).
A 40-μl PCR mix was prepared using a PCR Kit (Bioline, London, UK) containing each barcode-forward-primer set.
PCR reactions were performed in a Chromo4 thermocycler (BioRad) using a Multiplex PCR Mix (Qiagen).
Approximately 300 ng of chicken embryo DNA was used to analyze the engraftment of human MSCs by using a Universal PCR mix (Applied Biosystems, Foster City, CA) and primers and probes for a human-specific 850-bp fragment of the alpha-satellite DNA on human chromosome 17.
25 μl PCR volume reactions were set with 1 μl of each forward and reverse primers (0.5 μM final concentration) and using a 2× PCR mix from PROMEGA (Madison, USA).
For each PCR assay, two negative controls were used, i. e., sterile water and a PCR mix without DNA.
The cDNA was used as a template to perform qRT-PCR reactions (BioRad Laboratories, MyIQ PCR machine using SyBr Green PCR mix) using specific primer pairs (sequences available upon request).
PCR amplifications were performed using a commercial PCR master mix (kit QIAGEN Multiplex PCR) adding the DNA template (5 μl), 0.5 μM of each primer and water up to a final volume of 25 μl.
