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Fake positive colony in lamda RED reconbination, why?

About microscopic forms of life, including Bacteria, Archea, protozoans, algae and fungi. Topics relating to viruses, viroids and prions also belong here.

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Re:

Postby MXH » Thu Apr 08, 2010 11:13 am

JackBean wrote:I see. Do you have some positive control for experiment 2 with primers B?


No
Actually, I want knockout eight genes.
And I transform 16 PCR products (eight of them are amplified using PKD3 as templete, and others are amplified using PKD4) to 16 competent cell. I did this at least 5 times. None of them seems work. :(
I did every thing I can do, such as using different L-ara concentration to induce RED recombination enzymes, using different PCR product concentration to transform competent cell, using different drug (kanamycin or chloramphenicol) concentration.

The results are still "I can get several dozen colonies from plate with kanamycin, but cann't get any colony from chloramphenicol.
" and "I cann't amplify anti-kanamycin gene from these anti-kanamycin strain."


I need suggestionssss......
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Postby JackBean » Thu Apr 08, 2010 12:00 pm

OK, take your plasmid, which you use for transformation and try the PCR to see, whether your primers B work with your conditions ;)

EDIT
BTW in biology we use false positive, not fake ;)
http://www.biolib.cz/en/main/

Cis or trans? That's what matters.
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Re:

Postby MXH » Fri Apr 09, 2010 1:01 am

JackBean wrote:OK, take your plasmid, which you use for transformation and try the PCR to see, whether your primers B work with your conditions ;)

EDIT
BTW in biology we use false positive, not fake ;)



Your "BTW" is very constructive. Thank you, JackBean
You are the "JackBean" to me :lol:
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