Erro-prone PCR
wenhua zhang
Tuesday, 07 July 2009 04:52 UTC
I have been frustrating that the full-length GPCRs can’t be expressed at high levels in S. cerevesiae and what is much worse may come to the mistargeting of them. none of 40 has been, to date, localized correctly on the plasm membrane after induction for expression.
so, error-prone PCR, to my knowledge, may be an alternative to get the positive results through screenning.
ANYONE who has the high efficiency Erro-prone PCR protocol should win my gratitude as well as others.
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Replies
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Thank you for participating on our forum:
There are two protocols that we have in our system that require error-prone PCR.
The first uses a kit: Stratagene GeneMorph II random mutagenesis kit (Stratagene seem to do a series of kits that might be useful…)
The second gives the following advice:
Mutagenesis of AAV cap gene: Use the following error-prone PCR reaction protocol to mutate the entire cap sequence: 1 cycle of 5 min at 95 °C, 24 cycles of 30 s at 95 °C, 30 s at 62.5 °C, 2.5 min at 72 °C and finally 1 cycle of 10 min at 72 °C.
TableMutagenic buffer 70mM MgCl2, 500 mM KCl, 100 mM Tris (pH 8.3), 0.1% (wt/vol) gelatin. Combine 70 ul of 1 M MgCl2 (20.3 g MgCl26H2O per 100 ml deionized H2O), 500 ul of 1 M KCl (7.46 g KCl per 100 ml deionized H2O), 100 ul of 1 M Tris (pH 8.3; 12.1 g Tris base per 100 ml deionized H2O), 100 ul of 1% wt/vol gelatin (1 g gelatin per 100 ml deionized H2O) with deionized H2O to a final volume of 1 ml. Store at – 20 °C.
To cite the latter, assuming that it is at all helpful, follow this link
Best wishes with your research.
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