Team:Groningen/27 June 2013

From 2013.igem.org

Today the PCR products of the signal sequences (FliZ, EstA, MotB and LytB) are purified using gel purification. FliZ 9,2 ng/ul LytB 8,2 ng/ul MotB 8,8 ng/ul EstA 8,8 ng/ul

A PCR is done for some different silk constructs to start with. The following combinations are made (same protocol as 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. The bands appeared. A big smear is seen for all the silk products with a bigger band around 200 bp.