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