Table 2 Amplimers and PCR conditions.

From: Analysis of the frequency of single nucleotide polymorphisms in cytokine genes in patients with New Onset Diabetes After Transplant

(A) IFNG and TGFB

Amplimers

IFNG-FP generic:5′-tcaacaaagctgatactcca-3’

 

IFNG-RP A-allele:5′-ttcttacacaaaatcaaatca-3′;

IFNG-RP T-allele: 5′-ttcttacaacacaaaatcaaatct-3’

TGFB-FP generic: 5′-tccgtgggatactgagacac-3'

 

TGFB-RP C-allele: 5′-gcagcggtagcagcagcg-3′;

TGFB-RP T-allele: 5′-agcagcggtagcagcagca-3'

(B) IFNG and TGFB PCR conditions

PCR mix − total volume20 µl

PCR steps

Temp (°C)

Time

Cycles

Denaturation

95

1 min

–

Denaturation

95

15 s

10 cycles

Annealing

62

40 s

Extension

72

40 s

Denaturation

95

1 min

20 cycles

Annealing

54

1 min

Extension

72

1 min

Extension

72

5 min

 

(C) IL-4 and IL-6 amplimers

IL-4 FP:5′-gttgtaatgcagtcctcc-3′

IL-4RP:5′-actaggcctgatacg-3′

IL-6 FP: 5′-ttgtcaagacatgccaaagtgc-3′

IL-6 RP:5′gggaaaatcccacatttgataa-3′

(D) IL-4 and IL-6 PCR conditions

PCR mix − total volume25 µl

PCR steps

Temp (°C)

Time

Cycles

Denaturation

95

1 min

–

Denaturation

95

1 min

30 cycles

Annealing

55

1 min

 

Extension

72

1 min

 

Extension

72

5 min

 
  1. Digestion of IL4 done by BSmFI 65 °C 15 min 0.1 µl of RE and 1 µl cutting buffer for 10 µl PCR sample.
  2. Digestion of IL6 done by NIaIII 37 °C 15 min 0.02 µl of RE and 1 µl cutting buffer for 10 µl PCR sample.
  3. List of amplimers and PCR conditions used to amplify the promoter cytokines of their categories.
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