Uppsala-Sweden/17 August 2009

Top10 competent cells, making
As our earlier competent cells weren't working very well we decided to make new ones and in the process switch to the seemingly superior Top10 cells.

A pipette tip of Invitrogen Top10 competent cells was picked and put to grow in 1 ml of SOB media at 37 for 2 h. The liquid was streaked onto one SOB plate and also 10 µl was plated using 3.5 mm glass beads onto another SOB plate. the plates were left to grow at room temperature over night. This as given by the TOP10 chemically competent cells protocol

Also CCMB80 buffer was prepared as given by above protocol with the deviation that all ingredients were mixed and autoclaved were after the MnCl2 was solved in 20 ml of dH2O and sterile filtered into the buffer and the volume adjusted to 500 ml with autoclaved dH2O.

The resulting buffer was completely colourless (no manganese precipitate) and had a pH of 6.2.

This method yields a seemingly identical product to that of the protocol without necessitating sterile filtration of the whole volume, an extremely arduous task if one's only got access to 30 ml syringe filters.

--Anders 19:34, 17 August 2009 (UTC)

Phusion PCR of pirA with BB primers
I tried to BioBrick the pirA gene with the BBa-primers we designed for pirA earlier with the new pirA template, thus excluding genomic interference.

The first 5 rounds at 56,1°C annealing temperature.

The following 30 rounds at 70°C annealing temperature.

Unfortunately the PCR did not work.

--Karl.brune 15:44, 19 August 2009 (UTC)

Gel of Phusion PCR of pirA with BB primers


--Karl.brune 15:44, 19 August 2009 (UTC)