Lab 208

Main purpose

• Add USER endings to HAO gene extracted from Nitrosomonas europea

Who was in the lab

mysterious people

Procedure

Repeat HAO USER PCR from yesterday with same conditions and programs

Template: HAO extraction fragment -isolated from Nitrosomonas europea- (purified from PCR run on 04.09)

Primers: 18a & 18b

Two versions, one with 5% DMSO and one with 3% DMSO and two programs: Touchdown (62C -> 55) and a single annealing temperature of 59C.

PCR reaction master mix

• Add 49 ul of the master mix, and
• Add 1 ul DMSO (for the 5%) or 1 ul of MQ (for the 3%)

User reaction

was performed with HAO User fragment that was purified yesterday (but had low concentration) and pZA21::ara (promoter from the Biobrick K808000) and pZA21 with a tight arabinose promoter (from our SPL). Also Nir 1 User and Nir 2 User were ligated into the pZA21 ara tight for Nir. Negative controls were made for all three backbones.

Results

Gel 1

Gel of yesterday's PCR was empty (we lost the picture though...)

PCR was redone.

Gel 2

For the PCR results of today.

• 2-log ladder
• HAO User on 59C, 3% DMSO
• HAO User on 59C, 5% DMSO
• 2-log ladder

Gel 3

• 2-log ladder
• HAO User on touchdown, 3% DMSO
• HAO User on touchdown, 5% DMSO
• 2-log ladder

Conclusion

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