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From 2012.igem.org

week 6: 4 june - 10 june

Office work

Wiki page

Monday:

• Update of notebook and protocols
• Start to design the master-plan for the wiki page
• Designing the wiki template further

This monday we've worked further on updating the wiki, we are now on schedule with the notebook and protocols. We also made a master plan on how the wiki should look like. The first steps are being made to create a prize winning wiki page.

Tuesday:

• Update of notebook and protocols
• Designing the master-plan for the wiki further
• Creating the wiki homepage

Wednesday:

• Update of notebook and protocols
• Changing every Wensday into Wednesday

Thursday:

• Update notebook and protocols

Munich

Monday:

• Review of the two abstracts

Tuesday:

• Further editing the two abstracts
• Submitting the abstracts

Other

Tuesday:

• Start designing the WageningenUR clothing

[Meeting]

written by: Mark

Lab work

CCMV Bricking:

Monday:

• Check agar plates for colonies
• Grow colonies in LB medium + kanamycin, checked optical density
• Colonies:

1. CCMV-1
2. CCMV-2
3. d26-CCMV-1
4. d26-CCMV-2
5. d26-CCMV-3

• overnight in shaker 37 degree Celcius

The monday morning of this week started with a bit of a shock: our plates with our transformed E.coli we're almost completely empty. Luckely there was at least one colony on both of the plates with transformed E.coli containing bricks. We put them in Greiner tubes with LB-medium and a antibiotic and after a few hours checked their optical density. In the afternoon we prepared the samples for a PCR to validate the bricks.

Tuesday:

• Checked Greiner tubes
• Colonies:

1. CCMV-1: growth
2. CCMV-2: no growth
3. d26-CCMV-1: growth
4. d26-CCMV-2: growth
5. d26-CCMV-3: growth

• Mini prep the 4 remaining samples
• Prepare the mini prep samples for agarose gel
• Run the agarose gel (1% w/v agarose)
• Check agarose gel

The gel does not look promissing, as the size seems to be bigger than we expected. However, as we are dealing with plasmids, digestion will give the conclusive outcome, with linearized products on a gel. This will be done tomorrow

Wednesday:

• Minipreps of the 4 remaining samples is digested by Not1, for half an hour in a FastDigest Green buffer (fermentas)
• Digest products are ready to load on an agarose gel without adding loading buffer due to the Green buffer
• Run the agarose gel (1% w/v agarose)

The digestion products were not the expected products. We suspect that the linearized backbone pSB1K3 ligated with itself (suffix to suffix, prefix to prefix), as we get a single result arround 2.2k bp. Conclusion: the bricking failed.

Thursday:

CCMV test protocol:

Monday:

• Prepared two samples for dialysis according to the dialysis protocol
• Change: instead of sonicator for cell lysis, French press was used for one of the samples

We've also made two samples of wt CCMV VLP ready for dialysis. There is only one modification applied in the protocol, instead of using the horrible sonicator, we used the French press, which is a great apparatus and easy to use. In the late afternoon we started with the dialysis.

Tuesday:

• Changed the buffer 3 times according to the dialysis protocol
• Made an appointment for DLS

Wednesday:

• Changed the buffer 3 times according to the dialysis protocol

Thursday:

• Stopped dialysis
• Prepare samples for ultracentrifuge according to protocol
• Run ultracentrifuge, 4 degree Celcius 3 hours
• prepare samples for DLS

written by: Mark and Jeroen

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