Team:Warsaw/Calendar-Main/11 July 2008

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(Difference between revisions)
Line 13: Line 13:
<p>'''Preparation of construct omega-A'''<br>
<p>'''Preparation of construct omega-A'''<br>
<br>
<br>
-
1. PCR A in 50 ul<br>
+
1. PCR A in 50 µl<br>
-
template DNA - pKS-A4 1ul<br>
+
template DNA - pKS-A4 1 µl<br>
-
primer APNot - 2 ul<br>
+
primer APNot - 2 µl<br>
-
primer ALhomo2 - 2 ul<br>
+
primer ALhomo2 - 2 µl<br>
-
Pfu polymerase buffer + Mg2+ (from Fermentas) - 5 ul<br>
+
Pfu polymerase buffer + Mg2+ (from Fermentas) - 5 µl<br>
-
dNTPs - 1 ul <br>
+
dNTPs - 1 µl <br>
-
Pfu turbo - 0.5 ul<br>
+
Pfu turbo - 0.5 µl<br>
-
H2o - 38.5 ul<br>
+
H2o - 38.5 µl<br>
<br>
<br>
-
program:
+
program:<br>
-
1. 95oC 3'<br>
+
1. 95&deg;C 3'<br>
-
2. 95oC 30"<br>
+
2. 95&deg;C 30"<br>
-
3.62oC 45"<br>
+
3.62&deg;C 45"<br>
-
4.72oC 45"<br>
+
4.72&deg;C 45"<br>
-
5.72oC 10'<br>
+
5.72&deg;C 10'<br>
-
6. keeping in 4oC<br>
+
6. keeping in 4&deg;C<br>
</p>
</p>
-
2. PCR omega in 50 ul<br>
+
2. PCR omega in 50 µl<br>
-
template DNA - pUC19 1ul<br>
+
template DNA - pUC19 1 µl<br>
-
primer OmegaLS - 2 ul<br>
+
primer OmegaLS - 2 µl<br>
-
primer AOmegaPli - 2 ul<br>
+
primer AOmegaPli - 2 µl<br>
-
Pfu polymerase buffer + Mg2+ (from Fermentas) - 5 ul<br>
+
Pfu polymerase buffer + Mg2+ (from Fermentas) - 5 µl<br>
-
dNTPs - 1 ul <br>
+
dNTPs - 1 µl <br>
-
Pfu turbo - 0.5 ul<br>
+
Pfu turbo - 0.5 µl<br>
-
H2o - 38.5 ul<br>
+
H2o - 38.5 µl<br>
program:
program:
-
1. 95oC 3'<br>
+
1. 95&deg;C 3'<br>
-
2. 95oC 30"<br>
+
2. 95&deg;C 30"<br>
-
3. 62oC 45"<br>
+
3. 62&deg;C 45"<br>
-
4. 72oC 45"<br>
+
4. 72&deg;C 45"<br>
-
5. 72oC 10'<br>
+
5. 72&deg;C 10'<br>
-
6. keeping in 4oC<br>
+
6. keeping in 4&deg;C<br>
25 cycles
25 cycles

Revision as of 17:01, 26 September 2008

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Preparation of constructs with OmpA protein fusions
1. Colony PCR on colonies from plates with transformations OmpA_alpha.
2. Confirmed transformant colonies inoculated to liquid LB with kanamycin.
Cloning of protein Z DNA to OmpA constructs
1. 2 colonies was inoculated to liquid LB broth with kanamycin

Preparation of construct omega-A

1. PCR A in 50 µl
template DNA - pKS-A4 1 µl
primer APNot - 2 µl
primer ALhomo2 - 2 µl
Pfu polymerase buffer + Mg2+ (from Fermentas) - 5 µl
dNTPs - 1 µl
Pfu turbo - 0.5 µl
H2o - 38.5 µl

program:
1. 95°C 3'
2. 95°C 30"
3.62°C 45"
4.72°C 45"
5.72°C 10'
6. keeping in 4°C

2. PCR omega in 50 µl
template DNA - pUC19 1 µl
primer OmegaLS - 2 µl
primer AOmegaPli - 2 µl
Pfu polymerase buffer + Mg2+ (from Fermentas) - 5 µl
dNTPs - 1 µl
Pfu turbo - 0.5 µl
H2o - 38.5 µl

program: 1. 95°C 3'
2. 95°C 30"
3. 62°C 45"
4. 72°C 45"
5. 72°C 10'
6. keeping in 4°C
25 cycles

3. gel electrophoresis

4.reisolation from agarose gel