Team:Cambridge/Diary/Week 10

Monday

A few more people went on holiday and a few came back. Tom ran the gel for the lux-mOrange halves and nothing came out- he discovered that a few fragments we got out during the hectic Thursday and Friday are not of the right size, which might explain why our things are not working. That includes one part of the Ratiometrica vector backbone (vecB), and one part of the lux-mOrange backbone (vecA1). He re-PCRed these using touch-down PCR, and some of it seems to work now so we should start our Gibson again soon...

Tuesday

More PCR, more gels for Tom as he tries to get the correct fragments... He also grew up some overnight cultures with sfGFP-ampR plasmids and will miniprep so we now have more positive control templates.

Wednesday

Emmy did some reorganising and relabelling of all the tubes we've got as there are now too many tubes of DNA fragments for Ratiometrica and lux-mOrage! Tom did some more gel extractions and miniprepped the sfGFP-ampR out of the O/N cultures so now we have positive control again.

LUX-MORANGE PLAN OF ATTACK:

1) 2 x Two-piece Gibson to join the quarters of vector backbone together, and the mOrange and the other half of the vector (re-attempted with correct size fragment)

2a) PCR out each half (Touch-down PCR done... there is severe mispriming and the bands that we want is very very faint, not visible even on some lanes of the triplicates...)

2b) At the same time, attempt a 4-piece Gibson (done, will know tomorrow)

3) Two-piece Gibson the two halves

RATIOMETRICA PLAN OF ATTACK:

1) 2 x Two-piece Gibson to join each of the smaller fragments to the adjacent half of the vector backbone. (re-attempted one of the reactions with correct size fragment)

2a) PCR out each half (only one half came out last Friday, the other half is still not coming out even with the size correction and touch-down)

2b) At the same time, attempt a three-piece Gibson with the half from last Friday, and the two other fragments that are refusing to come out from Gibson (done, will know tomorrow)

3) Two-piece Gibson the two halves

Thursday

Tom re-PCRed a quarter of the vector backbone for Ratiometrica, because we suspect that the size of it from previous PCR was a bit dodgy. We will know if we get it of the right size later tonight.

Nothing's grown on the plates from last night- not even if the positive control. It might've been a problem with the Gibson protocol. We also tried to carry out gel electrophoresis directly with the Gibson products from yesterday night, unfortunately there are no bands. It might be because there were too little DNA, or it might just be the Gibson not working.

We have devised a new strategy to tackle the problem with Gibsoning Ratiometrica. We will be gibsoning together the smaller fragments later tonight, and then attempting a three-piece Gibson with the new fragment and also the 2 backbone fragments- we will know by tomorrow morning if this work. Also, we are ordering primers to ligate the vector fragments into a closed circle, to be used as a better positive control compared to the sfGFP-pSB4K5 that we have been using. Furthermore we will be looking into standard assembly as a plan B to get Ratiometrica out.

Happy news: Oli has colony PCR-ed out the Mg Riboswitch successfully, so he will be Gibsoning tonight and we will know if it all works tomorrow morning.

Friday