Team:HokkaidoU Japan/Notebook/plastic Week 5

Transformation

Transformation of pGEM into BL21. This transformation is the bioplastic production test in BL21.

1. Added 1ul of plasmid DNA to 50ul of thawed competent cells on ice.
2. Incubated on ice for 30min.
3. Added 600ul of LB.
4. Prepared and Labeled two petri dishes with LBA.
5. Plate 300ul of the transformation onto LBA dish and spread.
6. Added 900ul of LB to 100ul of the transformation and plated 300ul of it onto LBA dish then spread.
7. Incubated the plates at 37C for 15hrs 30min(22:00~13:30).

Liquid culture

Liquid culture of Transformed BL21(DE3)

1. Added 2ml of LBA into culture tubes.
2. Resuspended 2 colonies.
3. Incubated the tubes at 37C for OOhrs.

PHB production test

We prepared those three medium tubes and cultivated them in OOhrs.

1. 700ml LB + 0.7ul Amp
2. 700ml LB + 0.7ul Amp + 28ul 50% glucose
3. 700ml LB + 0.7ul Amp + 28ul 50% glucose + 1.4ul Sodium pantothenate

• BL21(DE3) = No.1~3 cultivated
• JM109 = No.2 only cultivated

Today the requested parts to HQ were sent! Parts were sent as agar stabs. We note here how to use these agar stabs.

Using agar stab(http://partsregistry.org/Help:Requesting_Parts)

We send out our part requests as agar stabs. The shelf life of these are short, so it is usually best to plate from the stab as soon as possible.

The agar should already have a hole from when it was stabbed. With an inoculating loop dipped into the stab, you can plate directly onto a petri dish with the appropriate antibiotic. Incubate the dish overnight at 37C (14-16hr) Pick a single colony to start up a culture Miniprep to extract plasmid DNA Use the part!