Team:Groningen/27 June 2013

From 2013.igem.org

(Difference between revisions)
(Created page with "Today the PCR products of the signal sequences (FliZ, EstA, MotB and LytB) are purified using gel purification. A PCR is done for some different silk constructs to start with. ...")
 
(9 intermediate revisions not shown)
Line 1: Line 1:
-
Today the PCR products of the signal sequences (FliZ, EstA, MotB and LytB) are purified using gel purification.  
+
'''Mirjam'''
 +
<br/>Today the PCR products of the signal sequences (FliZ, EstA, MotB and LytB) are purified using gel purification (Roche gel purification kit).
 +
<br/>FliZ  9,2 ng/ul
 +
<br/>LytB  8,2 ng/ul
 +
<br/>MotB  8,8 ng/ul
 +
<br/>EstA  8,8 ng/ul
-
A PCR is done for some different silk constructs to start with. The following combinations are made:
+
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):
<br/>1) signal sequence - N-terminal strep tag - silk
<br/>1) signal sequence - N-terminal strep tag - silk
<br/>2) signal sequence - silk - C-terminal strep tag
<br/>2) signal sequence - silk - C-terminal strep tag
<br/>3) signal sequence - silk - no strep tag
<br/>3) signal sequence - silk - no strep tag
<br/>4) silk without tag
<br/>4) silk without tag
 +
 +
The following protocol is used:
 +
<br/>98°C, 98°C, 50°C, 72°C, 72°C, 4°C
 +
<br/>0:30  0:10  0:25  0:27  10:00 forever
 +
 +
Added 1 ul serva/100 ml 0.8% agarose gel.
 +
<br/>Gel run at 90V for 22 min at a 0.8% agarose gel.
 +
<br/>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.

Latest revision as of 11:08, 28 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.