Lab 208

Main purpose

• Gradient PCR for Nir2 USER
• BioBrick construction
• SPL project
• Preparation for sequencing

Who was in the lab

Kristian, Julia, Henrike

Procedure

Gradient PCR for Nir2 USER

Set up new gradient PCR for Nir2 USER to run in 223. Reaction mix without MgCl2 but using DMSO and GC-buffer.

• template: Nir2 extraction fragment
• primers: 40a, 40b
• additives: 5% DMSO
• buffer: GC

Gradient went from 60C to 72C in 12 steps.

PCR for BioBrick construction

PCR amplified the constructs from Hello World and cytochromes and pSB1C3, which will be ligated together into biobricks.

constructs:

• GC-buffer, 5%DMSO, 2uL 50mM MgCl2
• primers: 53a, 53b
• templates: TAT3-2, Sec2, cycAX

Tried two different annealing temperatures. Program:

Note: Redid this PCR with X7 instead of PHUSION polymerase.

pSB1C3:

Done with primer pair 54.

SPL project

Picked single colonies of araBAD ref, constitutive SPL and constitutive ref transformants and replated them on plates sprayed with arabinose to prepare for Biolector experiment to measure the strength of the expression.

Preparation for sequencing

Diluted miniprepped plasmids down to 100 ng/uL. Have to redo cycAx in pZA21 purification because it was diluted too much.

Results

Gel on today's PCR for the Biobrick project

• 1 kb ladder
• Sec2 on 60C
• TAT3-2 on 60C
• cycAX on 60C
• Sec2 on 56C
• TAT3-2 on 56C
• cycAX on 56C
• negative for construct parts
• pSB1C3 backbone
• pSB1C3 backbone
• pSB1C3 backbone
• pSB1C3 backbone
• pSB1C3 backbone
• pSB1C3 backbone
• 1 kb ladder

Gel on screening PCR to amplify Nir2 USER

• 1 kb ladder
• Nir2 USER, sample 1
• Nir2 USER, sample 2
• Nir2 USER, sample 3
• Nir2 USER, sample 4
• Nir2 USER, sample 5
• Nir2 USER, sample 6
• Nir2 USER, sample 7
• Nir2 USER, sample 8
• Nir2 USER, sample 9
• Nir2 USER, sample 10
• Nir2 USER, sample 11
• Nir2 USER, sample 12
• 1 kb ladder

Samples 5-10 and 12 were cut out for gel purification.

Conclusion

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