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From 2008.igem.org

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Revision as of 21:05, 29 July 2008

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Contents 1 Construction of BioBrick intermediates 2 Methods 2.1 Attempt #1 2.1.1 Restriction digest 2.1.2 Gel purification 2.1.3 Ligated parts 2.1.4 Subcloned in DH5α 2.1.5 Colony PCR verification of constructs 2.2 Attempt #2 2.2.1 Restriction digest 2.2.2 Gel purification 2.2.3 Ligated parts 2.2.4 Subcloned in DB3.1 3 Results 3.1 Attempt #1 4 Discussion

Construction of BioBrick intermediates

• Construct:

• nir + rbs
• GFP + tt
• GFPf + tt

Methods

Attempt #1

Restriction digest

• Digested nir, GFP, and GFPf with EcoRI and SpeI in NEBuffer 2
• Digested B0034 (rbs) and B0024 (tt) with EcoRI and XbaI in NEBuffer 2 with BSA

Gel purification

• Ran digest reaction on an EtBr stained 2% gel to extract bands of interest

Ligated parts

• Ligated parts in 20 mu;l reactions:

• 4 μl B0034 (8.8 ng) + 2.42 μl nir (26.378 ng)
• 1 μl B0024 (15.5. ng) + 8 μl GFP (41.6 ng)
• 0.4 μl B0024 (6.2 ng) + 1.8 μl GFPf (1.8 ng)

Subcloned in DH5α

• Used 5 μl ligation reaction to transform
• Incubated at 37C with shaking for 100 min.

Colony PCR verification of constructs

Attempt #2

Restriction digest

• Digested nir, GFP, and GFPf with EcoRI and SpeI in NEBuffer EcoRI with BSA
• Sequentially digested B0034 (rbs) and B0024 (tt) with XbaI and EcoRI in their respective buffers

Gel purification

• Ran digest reaction on an EtBr stained 1.2% gel to extract bands of interest

Ligated parts

• Ligated parts in 20 mu;l reactions:

Subcloned in DB3.1

• Used 5 μl ligation reaction to transform

Results

Attempt #1

Discussion

• What was learned and how to do future experiments differently.

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