Lab 208

Main purpose

• Plasmid isolation of CycAX, TAT3-2, TAT3-1a, Sec2, HAO, pZA21::RFP and pZA21::araBAD::RFP.
• Nir2 with USER primers
• Prepare araBAD vector for inserts
• Prepare "Hello World" constructs for biobrick integration

Who was in the lab

Ariadni, Helen, Kristian, Julia

Procedure

Plasmid isolation

Plasmid have been isolated following the protocol provided by ORIGENE PowerPrep HP Plasmid Miniprep Kit.

Glycerol stock cultures

-80C stock solution of Sec2, TAT3-1a, TAT3-2

DpnI treatment

DpnI treated the USER fragments for the SPl project since there were many colonies on the negative controls which can only arise from template DNA used in the PCR. This is removed by DpnI.

User ligation and transformation

Repeated ligation and transformation from yesterday with same reaction mix to get seperated colonies useable for the SPL project. Plating in a gradient: 5uL, 50 uL, 100uL

SPL project

Picked single colonies of araBAD SPL transformants and replated them to prepare for Biolector experiment to measure the strength of the expression.

Gradient PCR on Nir2

Made gradient PCR on Nir2 to get the last USER fragment. The gradient was going from 60C → 72C with 12 reactions. Reactions where all made with same composition; GC-buffer, 2uL 50mM MgCl2 per reaction and 5% DMSO.

PCR on pSB1C3

Done with primerpair 54 as a two step PCR program with 98C in 10 sec. and 72C for 1:10 min looping 35 times.

PCR on constructs for biobrick integration

Done with primerpair 53 for all three templates; Sec, TAT3 and cycAX constructs. They where run on same PCR program with 56C annealing for 1 min. and 40 sec. extension.

Results

gel

• 1 kb ladder
• purification of the extraction fragment of Nir2
• Nir2 USER touchdown, sample 1
• Nir2 USER touchdown, sample 2
• Nir2 USER touchdown, sample 3
• Nir2 USER touchdown, sample 4
• Nir2 USER touchdown, sample 5
• Nir2 USER touchdown negative
• 1 kb ladder

The purification has the wrong length for Nir2 and was therefore discarded.

transformation

Yesterday's transformation yielded too many colonies that were inseparable, it will therefore be repeated and less volume will be plated.

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