Team:Groningen/Labwork/27 June 2013

Mirjam

Today the PCR products of the signal sequences (FliZ, EstA, MotB and LytB) are purified using gel purification (Roche gel purification kit).
FliZ 9,2 ng/ul
LytB 8,2 ng/ul
MotB 8,8 ng/ul
EstA 8,8 ng/ul Because of the low concentrations a new PCR is done using the PCR products. A PCR is done for some different silk constructs to start with. The following combinations are made (PCR mix preparation is done with the same protocol as described on 26-06-2013):
1) signal sequence - N-terminal strep tag - silk
2) signal sequence - silk - C-terminal strep tag
3) signal sequence - silk - no strep tag
4) silk without tag The following protocol is used:
98°C, 98°C, 50°C, 72°C, 72°C, 4°C
0:30 0:10 0:25 0:27 10:00 forever Added 1 ul serva/100 ml 0.8% agarose gel.
Gel run at 90V for 22 min at a 0.8% agarose gel.
Gel imaging revealed that no bands where present. Therefore it is placed at a high concentration of Ethidium Bromide and it is examined that the bands appear. A big smear is seen for all the silk products with a bigger band around 200 bp. Because of these results it is decided to do a gradient PCR on the first combination from 55-75 degrees Celsius.

Claudio

Based on the following bibliography:

Thermal Regulation of Membrane Lipid Fluidity by a Two-Component System in Bacillus subtilis by L. M. Bredeston§†, D. Marciano§, D. Albanesi¶, D. De Mendoza¶, and J. M. Delfino§

The three adaptation systems of Bacillus subtilis chemotaxis by Christopher V. Rao, George D. Glekas and George W. Ordal