Table 4.

Al-Quds University 16S general primers new long primers

Primer	PCR long primers	Oligomer: location	Tm (ºC)
QUGP-Fn3	5′-CAGGATTAGATACCCTGGTAGTCC-3′	24: 744–768	65
QUGP-Fn5	5′-ACTCCTACGGGAGGCAGCAG-3′	20: 323–343	65
QUGP-Fn6	5′-CCAGCAGCCGCGGTAATAC-3′	19: 479–497	62
QUGP-Rn1	5′-GGCTACCTTGTTACGACTTC-3′	20: 1471–1468	58
QUGP-Rn2	5′-TGACGGGCGGTGTGTACAAG-3′	20: 1406–1386	63
QUGP-Rn3	5′-GGCGTGGACTACCAGGGTATC-3′	21: 775–752	65

Long form QUGP were used to prepare golden mixtures

Underlined sequences, indicate the short QUGP prior to being re-designed. QUGP (underlined sequences) were extended mostly at their 5′-ends with conserved nucleotides, QUGP-F4/R4 were not modified since their Tm was 59.5°C and are flanked by variable nucleotides. The main differences between the QUGP and redesigned primers are limited to increased primer length and Tm which allowed annealing to be performed at 58°C instead of 50°C