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From 2012.igem.org

week 16: 13 august - 19 august

Office work

Lab work

TuYV

• Last week's 'brick' minipreps were PCR checked for the correct insert. Results were unclear at first, but a second check gave positive results for the TuYV Coat Protein brick, the CP + Histidine tag brick, and the CP + readthrough brick! Only the the CP + readthrough + His-tag brick wasn't found.

Hepatitis B

• 13 August (Lisa, Kees)

• SDS PAGE and VLP production

The cells (pre-induction and induced) were lysed using French press and analysed using SDS PAGE. A clear induction was shown, but because of a poor ladder, the HepB subunits could not be pointed out.

VLP production is continued according to the Hepatitis B VLP formation protocol [link]. We designed this protocol based on literature and the CCMV protocol.

• 16 August (Lisa, Kees)

• Finishing VLP formation

The dialysis was finished yesterday. Today, the dialysed solution was ultra-centrifuged, after which we dissolved the pellet in Formation Buffer, as little as we could use, about 0.2mL per centrifugation tube. An aliquot of all the tubes was made and stored at 4˚C until EM conformation.

Hepatitis B outside modification

• 13-14 August (Kees)

• Step 4 with alternative PCR program

Step 1-3 were repeated on 13 and 14 August. The DNA was gel purified after each step, enhancing the wanted product, though not excluding wrong products. Step 4 was repeated with an annealing step before adding the primers. Protocol: 1: 98˚C; 2min 2: 98˚C; 30s 3: 67˚C; 30s 4: 72˚C; 30s 5× from step 2 Added primers while: 5: 72˚C; 10min 6: 98˚C; 30s 7: 60˚C; 30s 8: 72˚C; 40s 35× from step 6 9: 72˚C; 10min 10: 4˚C; ∞

A smear was the result. In the blue circle a possible positive result can be seen. These word are carefully chosen, because similar bands should show in lanes 2-4, in which they are absent. Therefore, we need an alternative approach in which first the annealing is done after which the primers are added for elongation. Apparently, this approach does not work. The two fragments do not anneal properly, maybe because the Tm from the whole overhang is too high, 68-74˚C depending on calculation method used. Or, the chance of this whole event occurring as planned is just too low.

• 16 August (Kees)

• Final attempt PCR step 4

The final attempt was done using gel purified pcr products. This did not yield a better result than before. Therefore, a whole new strategy needs to be created.

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