Team:Warsaw/Calendar-Main/2 September 2008

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(New page: {{WarNotebook}} <!-- do not edit above me! --> Oczyszczanie białek: A-alfa, Z-alfa i Z-omega Piotr zaszczepienie A-alfa, Z-alfa i Z-omega z chodowli nocnej do świeżego LB, chodowa...)
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zaszczepienie A-alfa, Z-alfa i Z-omega z chodowli nocnej do świeżego LB, chodowanie do OD=0,5 indukcja rużnymi stężeniami IPG w 22 i 37C - pobranie próbek po 3h i następnego dnia rano (próbki zwirowano, odżucono pożywkę i zamrożono)
zaszczepienie A-alfa, Z-alfa i Z-omega z chodowli nocnej do świeżego LB, chodowanie do OD=0,5 indukcja rużnymi stężeniami IPG w 22 i 37C - pobranie próbek po 3h i następnego dnia rano (próbki zwirowano, odżucono pożywkę i zamrożono)
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<h3>Mutagenesis of protein A<br>Paweł</h3>
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<p>Mutagenesis of protein A was performed using 2 pairs of primers: <a href=https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/primers#ADelL+KpnI>ADelL+KpnI</a> and <a href=https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/primers#ADelP>ADelP</a> (deletion of aminoacids involved in interaction with protein Z) and <a href=https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/primers#AmutL+KpnI>AMutL+KpnI</a>  and <a href=https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/primers#AmutP>AMutP</a> (changing of few aminoacids involved in interaction with protein Z).</p><br>
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<p>Mutagenesis were performed on 3 vectors:  pACYClac+ompA-A-omega, pACYClac+ompa-A-alfa and pACYClac+ ompa-alfa-A.</p>
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<p>Each mutagenesis were performed using each primer at final concentration 0,1 uM, dNTPs at final concentration 0,25 uM and Walk (Pwo) polymerase (shipped by <a href=http://aabiot.com/>A&A Biotechnology</a>), with 100 ng of DNA template. </p>
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<p><br>PCR program (15 cycles):
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<pre style="text-align: left">94C 5 min
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94C 30 s
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55C 30 s
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72C 10 min
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72C 8 min
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4C pause
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</pre></p>
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Revision as of 21:54, 5 October 2008

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Oczyszczanie białek: A-alfa, Z-alfa i Z-omega Piotr zaszczepienie A-alfa, Z-alfa i Z-omega z chodowli nocnej do świeżego LB, chodowanie do OD=0,5 indukcja rużnymi stężeniami IPG w 22 i 37C - pobranie próbek po 3h i następnego dnia rano (próbki zwirowano, odżucono pożywkę i zamrożono)

Mutagenesis of protein A
Paweł

Mutagenesis of protein A was performed using 2 pairs of primers: ADelL+KpnI and ADelP (deletion of aminoacids involved in interaction with protein Z) and AMutL+KpnI and AMutP (changing of few aminoacids involved in interaction with protein Z).


Mutagenesis were performed on 3 vectors: pACYClac+ompA-A-omega, pACYClac+ompa-A-alfa and pACYClac+ ompa-alfa-A.

Each mutagenesis were performed using each primer at final concentration 0,1 uM, dNTPs at final concentration 0,25 uM and Walk (Pwo) polymerase (shipped by A&A Biotechnology), with 100 ng of DNA template.


PCR program (15 cycles): 

94C 5 min

94C 30 s
55C 30 s
72C 10 min

72C 8 min

4C pause

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