DNA was extracted from snap-frozen human PDO cell pellets by the Sample Processing Lab using an AllPrep kit (Qiagen). Primers sequences for amplification and sequencing of exons of the KRAS gene that contain the G12/13 codons are listed below:
KRAS G12/13 forward: 5′-CTGGTGGAGTATTTGATAGTG-3′
KRAS G12/13 reverse: 5′-CTGTATCAAAGAATGGTCCTG-3′.
The following PCR conditions were used as previously described54 (link) and specifically noted in that paper: ‘94 °C for 2 min; three cycles of 94 °C for 30 s, 64 °C for 30 s and 72 °C for 30 s; three cycles of 94 °C for 30 s, 61 °C for 30 s and 72 °C for 30 s; three cycles of 94 °C for 30 s, 58 °C for 30 s and 72 °C for 30 s and three cycles of 94 °C for 30 s, 57 °C for 30 s and 72 °C for 30 s, followed by 72 °C for 5 min and a hold at 4 °C’. PCR products were purified using a QIAquick PCR purification kit and sent to and sequenced by Eurofins. The resulting sequences were analyzed using Mutation Surveyor software (SoftGenetics).
KRAS G12/13 forward: 5′-CTGGTGGAGTATTTGATAGTG-3′
KRAS G12/13 reverse: 5′-CTGTATCAAAGAATGGTCCTG-3′.
The following PCR conditions were used as previously described54 (link) and specifically noted in that paper: ‘94 °C for 2 min; three cycles of 94 °C for 30 s, 64 °C for 30 s and 72 °C for 30 s; three cycles of 94 °C for 30 s, 61 °C for 30 s and 72 °C for 30 s; three cycles of 94 °C for 30 s, 58 °C for 30 s and 72 °C for 30 s and three cycles of 94 °C for 30 s, 57 °C for 30 s and 72 °C for 30 s, followed by 72 °C for 5 min and a hold at 4 °C’. PCR products were purified using a QIAquick PCR purification kit and sent to and sequenced by Eurofins. The resulting sequences were analyzed using Mutation Surveyor software (SoftGenetics).
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