Rehydration of Registry DNA

Experimental Details and Rationale

Registry DNA was rehydrated for completion of the Interlab Study. Also, Part BBa_K934001 (phaC1-A-B1) was rehydrated by the Secretion subgroup and transformed into our chassis so that P(3HB) was produced and preliminary secretion assays could be performed before the Synthesis subgroup had completed their cloning.

Materials

iGEM 2017 distribution kit

ddH₂O

Protocol

1. Add 10 μL of ddH₂O to the desired well.
2. Pipette up and down 3-5 times.
3. Incubate at room temperature for 10 minutes.
4. Transform cells with 1 μL of rehydrated DNA. Store the remaining amount at -20°C.

Rehydration of IDT Synthesized DNA

Experimental Details and Rationale

Our genetic parts were ordered from IDT and arrived as a dry, lyophilized powder. They were resuspended in aqueous solution for cloning into pSB1c3 or pET29B vectors and to ligate multiple parts together.

Materials

Synthesized DNA from IDT (gBlocks)

ddH₂O

Protocol

1. Centrifuge tube containing the synthesized DNA for 3-5 seconds at 3000g to ensure that all material is at the bottom of the tube.
2. Add ddH₂O to reach a final concentration of 50 ng/μL.
3. Vortex.
4. Incubate at 50°C for 20 minutes.
5. Briefly vortex and centrifuge . Store at -80°C.

Plasmid MiniPrep from Escherichia coli

Experimental Details and Rationale

coli DH5ɑ and BL21 were lysed and the pSB1c3 or pET29B vectors were isolated to be used in cloning our genetic constructs. Bacterial clones were lysed for analysis (eg: confirmation restriction digest, genetic sequencing).

Materials

2.5 mL overnight culture of bacteria in Luria-Bertani broth with appropriate buffer in 16x125mm culture tube

Resuspension buffer (stored at 4°C)

• 50 mM Tris-HCl, pH 8

• 10 mM EDTA

• 100 μg/mL RNase A

Lysis buffer

• 200 mM NaOH

• 1% (v/v) SDS

Precipitation buffer

• 3 M CH₃CO₂K, pH 5.5

Isopropanol

70% ethanol

Table-top centrifuge

Ice bucket

2-mL microcentrifuge tubes

1.5-mL microcentrifuge tubes

ddH₂O 3

Protocol

1. Step 1
2. Step 2
3. Step 3
4. etc.