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Team:Groningen/27 June 2013
From 2013.igem.org
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| - | 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/>FliZ 9,2 ng/ul | ||
<br/>LytB 8,2 ng/ul | <br/>LytB 8,2 ng/ul | ||
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Because of the low concentrations a new PCR is done using the PCR products. | 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 (same protocol as 26-06-2013): | + | 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 | ||
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Added 1 ul serva/100 ml 0.8% agarose gel. | 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 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 | + | <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.
