lab 208

Main purpose

Who was in the lab

Procedure

Colony PCR to verify Nir in pZA21::ara-tight

Redid colony PCR with sequencing primers instead. Two primer pairs to test for the two USER parts. Pair 1: Nir_FW_4_Seq + Nir_RV_5_Seq . Pair 2: Nir_FW_13_Seq + Nir_RV_14_Seq.

Used Q5 premix. Calculated annealing temperature for pair 1: 60C, pair 2: 64C

Note: The resulting gel showed a lot of bands that are consistent for all colonies. We repeated the PCR for one of the colonies with the following annealing temperatures: pair 1: 67C, pair 2: 68C

Results

Gel

• 1 kb ladder
• Nir colony 1, primer pair 1
• Nir colony 1, primer pair 2
• Nir colony 2, primer pair 1
• Nir colony 2, primer pair 2
• Nir colony 3, primer pair 1
• Nir colony 3, primer pair 2
• Nir colony 4, primer pair 1
• Nir colony 5, primer pair 2
• Nir colony 5, primer pair 1
• Nir colony 5, primer pair 2
• Nir colony 6, primer pair 1
• Nir colony 6, primer pair 2
• 1 kb ladder

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